Non-reducing saccharide-forming enzyme, trehalose-releasing enzyme, and process for producing saccharides using the enzymes

ABSTRACT

A non-reducing saccharide-forming enzyme and a trehalose-releasing enzyme, which have an optimum temperature in a medium temperature range, i.e., a temperature of over 40 or 45° C. but below 60° C.; and an optimum pH in an acid pH range, i.e., a pH of less than 7. The two-types of enzymes can be obtained in a desired amount, for example, by culturing in a nutrient culture medium microorganisms capable of producing the enzymes or by recombinant DNA technology.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of parent application Ser.No. 09/392,253, filed Sep. 9, 1999, now abandoned the entire contents ofwhich being hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a non-reducing saccharide-formingenzyme, a trehalose-releasing enzyme, and a process for producingsaccharides using the enzymes.

2. Description of the Prior Art

Trehalose is a disaccharide consisting of two moles of glucose bound attheir reducing residues, and is widely found in nature, for example, inmicroorganisms, fungi, algae, insects, Crustacea, etc. Since thesaccharide has long been known as a useful saccharide substantially freeof reducibility and having a satisfactory moisture-retaining action, ithas been expected to use in extensive fields including foods, cosmetics,and pharmaceuticals. However, no efficient production of the saccharidewas established, and this narrows the use of trehalose in spite of itsoutstanding expectation. Thus supply of trehalose in a lower cost isgreatly expected.

As a proposal for such an expectation, the present inventors had alreadyestablished a process for enzymatically producing trehalose frommaterial starches through their energetic studies. The process ischaracterized by a step of subjecting reducing partial starchhydrolysates to the action of a non-reducing saccharide-forming enzyme,which forms a non-reducing saccharide having a trehalose structure as anend unit from reducing partial starch hydrolysates, and to the action ofa trehalose-releasing enzyme which acts on a non-reducing saccharidehaving a trehalose structure as an end unit in order to hydrolyze andrelease trehalose from the rest of the non-reducing saccharide. Theseenzymes and processes thereof are disclosed in Japanese Patent KokaiNos. 143,876/95, 213,283/95, 322,883/95, 298,880/95, 66,187/96,66,188/96, 73,504/96, 84,586/96, and 336,388/96, applied for by the sameapplicant as the present invention. Thus, a low-cost production oftrehalose was attained.

During the studies, they found an original finding that the non-reducingsaccharide-forming enzyme can be applied for a novel production ofnon-reducing saccharides that can overcome conventional drawbackresiding in reducing partial starch hydrolysates. As a problem, reducingpartial starch hydrolysates such as dextrins and maltooligosaccharideshave advantageous features that they can be used as sweeteners andenergy-supplementing saccharide sources, but as a demerit they arehighly reactive with substances because of their reducibility and aresusceptible to browning reaction when coexisted with amino acids and/orproteins and to readily deteriorate their quality. To overcome such aproblem, it is only known a method to convert reducing partial starchhydrolysates into sugar alcohols using a high-pressure hydrogenationmethod, etc. In actual use, the method, however, needs much heats andinstruments constructed under consideration of safety in view of the useof hydrogen, resulting in a higher cost and much labor cost. On thecontrary, the aforesaid non-reducing saccharide-forming enzyme asmentioned previously acts on reducing partial starch hydrolysates andforms non-reducing saccharide having a trehalose structure as an endunit, and the reaction proceeds under a relatively-mild condition due toits enzymatic reaction. Using the action of the enzyme, the presentinventors established a novel efficient process for non-reducingsaccharides using the enzyme, that can overcome conventional drawbackresiding in reducing partial starch hydrolysates. Because of thesefindings, the development of applicable uses for trehalose andnon-reducing saccharides have become to be flourished in various fields,and this diversifies the uses of these saccharides and now remarkablyincreases the demands of the saccharides in a wide variety of fields.

Under these circumstances, a more efficient process for producingtrehalose and non-reducing saccharides having a trehalose structure hasbeen more expected in this art. A key to such an expectation is toestablish a non-reducing saccharide-forming enzyme and atrehalose-releasing enzyme with various optimum conditions, and toprovide a wide variety of sources for such enzymes usable in theproduction of the saccharides. Thus, an optimum enzyme can be chosenfrom various types of enzymes depending on the optimum conditions ofanother enzymes usable in combination with the above enzymes to producethe desired saccharides, as well as on installations and final uses ofthe saccharides produced, resulting in an efficient production of thesaccharides. Conventionally known non-reducing saccharide-formingenzymes can be grouped into those having optimum temperatures ofrelatively-lower temperatures of about 40° C. or lower, and those havingoptimum temperatures of relatively-higher temperatures of about 60° C.or higher. While conventionally known trehalose-releasing enzymes can begrouped into those having optimum temperatures in a relatively-lowertemperature range, about 45° C. or lower, and those having optimumtemperatures in a relatively-higher temperature range, about 60° C. orhigher. However, any non-reducing saccharide-forming enzyme and atrehalose-releasing enzyme having an optimum temperature in a mediumtemperature range, about 50° C., have never yet been opened.

Among saccharide-related enzymes used in the production of saccharidesfrom starch materials, enzymes as a major group have an optimumtemperature in a medium temperature range. Such enzymes may be requiredin the process for producing the aforesaid trehalose and non-reducingsaccharides; No non-reducing saccharide-forming enzyme and notrehalose-releasing enzyme, which have an optimum temperature in amedium temperature range, have not yet been established so that therehas not yet been realized a process for producing saccharides in asufficient yield using either or both of these enzymes together with theabove saccharide-related enzymes. Depending on installations forproducing saccharides and final uses of them, there have been requiredenzymes having an optimum temperature in a medium temperature range intheir enzymatic reactions. It is far from saying that it has establisheda process for producing saccharides in a satisfactorily-high yield usinga non-reducing saccharide-forming enzyme and a trehalose-releasingenzyme. As described above the establishment of a non-reducingsaccharide-forming enzyme and a trehalose-releasing enzyme having anoptimum temperature in a medium temperature range, and a process forproducing saccharides comprising non-reducing saccharides are in greatdemand.

SUMMARY OF THE INVENTION

In view of this, the first object of the present invention is to providea non-reducing saccharide-forming enzyme having an optimum temperaturein a medium temperature range.

The second object of the present invention is to provide a DNA encodingthe non-reducing saccharide-forming enzyme.

The third object of the present invention is to provide a process forproducing the non-reducing saccharide-forming enzyme.

The fourth object of the present invention is to provide atrehalose-releasing enzyme having an optimum temperature in a mediumtemperature range.

The fifth object of the present invention is to provide a DNA encodingthe trehalose-releasing enzyme.

The sixth object of the present invention is to provide a process forproducing the trehalose-releasing enzyme.

The seventh object of the present invention is to provide amicroorganism capable of producing the non-reducing saccharide-formingenzyme and/or the trehalose-releasing enzyme.

The eighth object of the present invention is to provide a process forproducing saccharides comprising non-reducing saccharides, which usesthe non-reducing saccharide-forming enzyme and/or thetrehalose-releasing enzyme.

In order to attain the above objects, the present inventors extensivelyscreened microorganisms, that can overcome the objects, in soils. As aresult, they found that a microorganism newly isolated from a soil inAko-shi, Hyogo, Japan, produced enzymes that can solve the aboveobjects. The present inventors isolated separatory the desirednon-reducing saccharide-forming enzyme and trehalose-releasing enzymefrom the microorganism, and then identified their properties, revealingthat the enzymes both had an optimum temperature in a medium temperaturerange. The identification of the microorganism confirmed that it was anovel microorganism of the genus Arthrobacter, and named Arthrobactersp. S34. The microorganism was deposited on Aug. 6, 1998, in theNational Institute of Bioscience and Human-Technology Agency ofIndustrial Science and Technology, Higashi 1-1-3, Tsukuba-shi, Ibaraki,Japan, and accepted and has been maintained by the institute under theaccession number of FERM BP-6450.

The present inventors continued studying, isolated DNAs encoding theabove-identified enzymes from the microorganism, Arthrobacter sp. S34,FERM BP-6450, decoded the nucleotide sequences, and determined the aminoacid sequences of the enzymes. The inventors confirmed that Arthrobactersp. S34, FERM BP-6450, and transformants, into which the DNAs obtainedin the above had been introduced in a usual manner, produced desiredamounts of enzymes. It was also confirmed that the enzymes thus obtainedcan be advantageously used in producing saccharides which comprisetrehalose and non-reducing saccharides having a trehalose structure in amedium temperature range. The present invention was made based on thesefindings.

The first object of the present invention is solved by a novelnon-reducing saccharide-forming enzyme that forms a non-reducingsaccharide having a trehalose structure as an end unit from reducingpartial starch hydrolysates, and has an optimum temperature in a mediumtemperature range.

The second object of the present invention is solved by a DNA encodingthe non-reducing saccharide-forming enzyme.

The third object of the present invention is solved by a process forproducing the non-reducing saccharide-forming enzyme, characterized inthat it comprises the steps of culturing a microorganism capable ofproducing the enzyme, and collecting the produced enzyme from theculture.

The fourth object of the present invention is solved by a noveltrehalose-releasing enzyme which specifically hydrolyses a non-reducingsaccharide having a trehalose structure as an end unit and a glucosepolymerization degree of at least 3 to release trehalose from the restof the non-reducing saccharide, and which has an optimum temperature ina medium temperature range.

The fifth object of the present invention is solved by a DNA encodingthe trehalose-releasing enzyme.

The sixth object of the present invention is solved by a process forproducing the trehalose-releasing enzyme, characterized in that itcomprises the steps of culturing a microorganism capable of producingthe enzyme, and collecting the produced enzyme from the culture.

The seventh object of the present invention is solved by a microorganismselected from Arthrobacter sp. S34, FERM BP-6450, and mutants thereof.

The eighth object of the present invention is solved by a process forproducing saccharides, comprising the steps of allowing the either orboth of the above enzymes to act on reducing partial starch hydrolysatesto produce non-reducing saccharides, and collecting the non-reducingsaccharides or saccharide compositions having a relatively-lowreducibility and containing the non-reducing saccharides.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1 is a figure that shows the influence of temperature on theactivity of a non-reducing saccharide-forming enzyme from Arthrobactersp. S34, FERM BP-6450, according to the present invention.

FIG. 2 is a figure that shows the influence of pH on the activity of anon-reducing saccharide-forming enzyme from Arthrobacter sp. S34, FERMBP-6450, according to the present invention.

FIG. 3 is a figure that shows the influence of temperature on thestability of a non-reducing saccharide-forming enzyme from Arthrobactersp. S34, FERM BP-6450, according to the present invention.

FIG. 4 is a figure that shows the influence of pH on the stability of anon-reducing saccharide-forming enzyme from Arthrobacter sp. S34, FERMBP-6450, according to the present invention.

FIG. 5 is a restriction map of the recombinant DNA pGY1 according to thepresent invention. The bold line shows the nucleotide sequence fromArthrobacter sp. S34, FERM BP-6450. The black arrow within the bold lineshows a nucleotide sequence encoding the present non-reducingsaccharide-forming enzyme, while the oblique arrow shows a nucleotidesequence encoding the present trehalose-releasing enzyme.

FIG. 6 is a restriction map of the recombinant DNA pGY2 according to thepresent invention. The bold line shows the nucleotide sequence fromArthrobacter sp. S34, FERM BP-6450. The black arrow within the bold lineshows a nucleotide sequence encoding the present non-reducingsaccharide-forming enzyme.

FIG. 7 is a restriction map of the recombinant DNA pGY3 according to thepresent invention. The black arrow shows the nucleotide sequence,encoding the present non-reducing saccharide-forming enzyme, fromArthrobacter sp. S34, FERM BP-6450.

FIG. 8 is a figure that shows the influence of temperature on theactivity of a trehalose-releasing enzyme from Arthrobacter sp. S34, FERMBP-6450, according to the present invention.

FIG. 9 is a figure that shows the influence of pH on the activity of atrehalose-releasing enzyme from Arthrobacter sp. S34, FERM BP-6450,according to the present invention.

FIG. 10 is a figure that shows the influence of temperature on thestability of a trehalose-releasing enzyme from Arthrobacter sp. S34,FERM BP-6450, according to the present invention.

FIG. 11 is a figure that shows the influence of pH on the stability of atrehalose-releasing enzyme from Arthrobacter sp. S34, FERM BP-6450,according to the present invention.

FIG. 12 is a restriction map of the recombinant DNA pGZ2 according tothe present invention. The bold line shows the nucleotide sequence fromArthrobacter sp. S34, FERM BP-6450. The oblique arrow within the boldline shows a nucleotide sequence encoding the presenttrehalose-releasing enzyme.

FIG. 13 is a restriction map of the recombinant DNA pGZ3 according tothe present invention. The oblique arrow shows the nucleotide sequencefrom Arthrobacter sp. S34, FERM BP-6450.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a non-reducing saccharide-formingenzyme and a trehalose-releasing enzyme, and a process for producing asaccharide using either or both of the enzymes. The wording“non-reducing saccharide-forming enzyme” as referred to in the presentinvention represents an enzyme which has an action of forming anon-reducing saccharide having a trehalose structure as an end unit fromreducing partial starch hydrolysates. The wording “trehalose-releasingenzyme” as referred to in the present invention represents an enzymewhich specifically hydrolyses a non-reducing saccharide having atrehalose structure as an end unit and a glucose polymerization degreeof at least 3 to release trehalose from the rest of the non-reducingsaccharide. The wording “a medium temperature range” as referred to inthe present invention represents a middle temperature range in reactiontemperatures which are conventionally used in producing saccharides fromstarch materials by an enzymatic reaction. In most cases of suchprocesses, different reaction temperatures of about 10° C. to about 100°C. and a round the temperatures are used. The nonreducingsaccharide-forming enzyme according to the present invention has anaction as such an enzyme and has an optimum temperature in a mediumtemperature range, preferably a temperature range over 40° C. but lessthan 60° C. and more preferably it has an optimum pH in an acid pH rangein addition to the optimum temperature. The trehalose-releasing enzymeaccording to the present invention has an action as such an enzyme andhas an optimum temperature in a medium temperature range, preferably atemperature range over 45° C. but below 60° C., and more preferably ithas an optimum pH in an acid pH range in addition to the optimumtemperature. These present enzymes should not be restricted to theirorigins and sources.

The activity of the present non-reducing saccharide-forming enzyme isassayed as follows: One ml of an enzyme solution is added to four ml of1.25 w/v % maltopentaose as a substrate in 20 mM phosphate buffer (pH6.0), and the mixture solution is incubated at 50° C. for 60 min. Thereaction mixture is heated at 100° C. for 10 min to suspend theenzymatic reaction, and the reaction mixture is precisely diluted by 10times with deionized water, followed by determining the reducing powerof the diluted solution on the Somogyi-Nelson's method. As a control, anenzyme solution, which had been heated at 100° C. for 10 min toinactivate the enzyme, is treated similarly as above. One unit activityof the present enzyme is defined as the amount of enzyme whicheliminates the reducing power of that of one μ mole of maltopentaose perminute when determined with the above-mentioned assay. The optimumtemperature of the enzyme as referred to in the present invention isdetermined in accordance with the assay; It is assayed by adjusting theenzymatic reaction temperature at different temperatures including 50°C., allowing a prescribed amount of the enzyme to act on the substrateat the different temperatures according to the assay, and determiningthe reduction level of reducing power at the temperatures in accordancewith the assay, followed by comparing the determined reduction levelsone another and determining the optimum temperature of the presentenzyme that showed a maximum temperature.

The activity of the present trehalose-releasing enzyme is assayed asfollows: One ml of an enzyme solution is added to four ml of 1.25 w/v %maltotriosyltrehalose, i.e., α-maltotetraosyl-α-D-glucoside, as asubstrate, in 20 mM phosphate buffer (pH 6.0), and the mixture solutionis incubated at 50° C. for 30 min, followed by suspending the enzymaticreaction by the addition of the Somogyi copper solution and assaying thereducing power by the Somogyi-Nelson's method. As a control, it issimilarly assayed using an enzyme solution which has been inactivated byheating at 100° C. for 10 min. One unit activity of the present enzymeis defined as the amount of enzyme which increases the reducing power ofone μ mole of glucose per minute when determined with theabove-mentioned assay. The optimum temperature of the enzyme as referredto in the present invention is determined in accordance with the assay;It is assayed by adjusting the enzymatic reaction temperature at thedifferent temperatures including 50° C., allowing a prescribed amount ofthe enzyme to act on the substrate at the temperatures according to theassay, and determining the increased level of reducing power at thedifferent temperatures in accordance with the assay, followed bycomparing the determined increased levels one another and determiningthe optimum temperature of the present enzyme that showed a maximumtemperature.

Explaining the present non-reducing saccharide-forming enzyme based onthe amino acid sequence, the enzyme has the amino acid sequence of SEQID NO: 1 as a whole, and has the amino acid sequences of SEQ ID NOs: 2to 6 as partial amino acid sequences in some cases. In addition to theseenzymes having the whole of the above-identified amino acid sequences,the present invention includes another types of enzymes which comprise apart of any one of the amino acid sequences selected therefrom or whichhave both the action as the present non-reducing saccharide-formingenzyme and the above-identified optimum temperature. Examples of theamino acid sequences of such enzymes are those which contain, within theamino acid sequences, a partial amino acid sequence or an amino acidresidue that are related to the expression of the properties of thepresent non-reducing saccharide-forming enzyme, and which one or moreamino acids are replaced with different amino acids, added thereuntoand/or deleted therefrom other than the above partial amino acidsequence or the amino acid residue. Examples of the amino acid sequencesreplaced with different amino acids as referred to in the presentinvention include those which less than 30% and preferably less than 20%of the amino acid sequences composing the amino acid sequence of SEQ IDNO: 1 are replaced with another amino acids which have similarproperties and structures to respective ones to be replaced. Examples ofgroups of such amino acids are a group of aspartic acid and glutamicacid as acid amino acids, one of lysine, arginine, and histidine asbasic amino acids, one of asparagine and glutamine as amid-type aminoacids, one of serine and threonine as hydroxyamino acids, and one ofvaline, leucine and isoleucine as branched-chain amino acids. Examplesof another amino acid sequences of the present enzyme containing a partof any one of the amino acid sequences selected from SEQ ID NOs: 1 to 6are those which might have a substantially similar stereo-structure tothe one of the amino acid sequence of SEQ ID NO: 1, i.e., replacement,deletion and/or addition of amino acid(s) are introduced into the aminoacid sequence of SEQ ID NO: 1. The stereo-structure of proteins isestimable by screening commercially available databases forstereo-structures of proteins which have amino acid sequences related tothe aiming ones and have revealed stereo-structures, referencing thescreened stereo-structures, and using commercially available soft waresfor visualizing stereo-structures. The above-identified amino acidsequence of the present non-reducing saccharide-forming enzyme has ahomology of at least 57%, preferably at least 70%, and more preferablyat least 80% to SEQ ID NO: 1.

As described above, the non-reducing saccharide-forming enzyme shouldnot be restricted to a specific origin/source. Examples of such arethose derived from microorganisms, i.e., those of the genusArthrobacter, Arthrobacter sp. S34, FERM BP-6450, and its mutants. Themutants can be obtained by treating in a usual manner Arthrobacter sp.S34, FERM BP-6450, with known mutagens such asN-methyl-N′-nitro-N-nitrosoguanidine, ethyl methanesulfonate,ultraviolet, and transposon; screening the desired mutants capable ofproducing a non-reducing saccharide-forming enzyme and having an optimumtemperature at temperatures in a medium temperature range, and usuallyat temperatures in the range of over 40° C. but below 60° C. The enzymefrom Arthrobacter sp. S34, FERM BP-6450, usually has the amino acidsequences of SEQ ID NOs: 1 to 6. Another non-reducing saccharide-formingenzymes from microorganisms of mutants Arthrobacter sp. S34, FERMBP-6450, and another microorganisms comprise the whole or a part of anyone of the amino acid sequences of SEQ ID NOs: 1 to 6. Concrete examplesof another enzymes include recombinant enzymes which act as the presentnon-reducing saccharide-forming enzyme and have an optimum temperatureat temperatures in a medium temperature range, and usually attemperatures of over 40° C. but below 60° C. The recombinant enzymes canbe obtainable by applying the recombinant DNA technology for the DNAencoding the present non-reducing saccharide-forming enzyme, and havethe whole or a part of any one of the amino acid sequences of SEQ IDNOs: 1 to 6.

Most of the non-reducing saccharide-forming enzyme according to thepresent invention has the following physicochemical properties:

-   -   (1) Action Forming a non-reducing saccharide having a trehalose        structure as an end unit from a reducing partial starch        hydrolysates having a degree of glucose polymerization of 3 or        higher;    -   (2) Molecular weight About 75,000±10,000 daltons on sodium        dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE);    -   (3) Isoelectric point (pI) About 4.5±0.5 on isoelectrophoresis        using ampholyte;    -   (4) Optimum temperature About 5° C. when incubated at pH 6.0 for        60 min;    -   (5) Optimum pH About 6.0 when incubated at 50° C. for 60 min;    -   (6) Thermal stability Stable up to a temperature of about 55° C.        when incubated at pH 7.0 for 60 min; and    -   (7) pH Stability Stable at pHs of about 5.0 to about 10.0 when        incubated at 4° C. for 24 hours.

The present non-reducing saccharide-forming enzyme can be obtained in aprescribed amount by the later described present process for producingthe same.

The present invention provides a DNA encoding the present non-reducingsaccharide-forming enzyme. Such a DNA is quite useful in producing theenzyme in the form of a recombinant protein. In general, the DNAincludes those which encode the enzyme independently of itsorigin/source. Examples of such a DNA are those which contain the wholeor a part of the nucleotide sequence of SEQ ID NO: 7 or complementaryones thereunto. The DNA comprising the whole of the nucleotide sequenceof SEQ ID NO: 7 encodes the amino acid sequence of SEQ ID NO: 1. TheDNAs, which contain the whole or a part of the nucleotide sequence ofSEQ ID NO: 7, include those which have an amino acid sequence relatingto the expression of the properties of the present non-reducingsaccharide-forming enzyme, and have a nucleotide sequence correspondingto the amino acid sequence, and the nucleotide sequence of SEQ ID NO: 7introduced with a replacement, deletion and/or addition of one or morebases while retaining the nucleotide sequence relating to the expressionof the properties of the present non-reducing saccharide-forming enzyme.The DNAs according to the present invention should include those whichone or more bases are replaced with different ones based on thedegeneracy of genetic code. Also the DNAs according to the presentinvention include those which comprise the nucleotide sequences thatencode the present non-reducing saccharide-forming enzyme and furthercomprise additional one or more another nucleotide sequences selectedfrom the group consisting of ribosome-binding sequences such as aninitiation codon, termination codon, and Shine-Dalgarno sequence;nucleotide sequences encoding signal peptides, recognition sequences forappropriate restriction enzymes; nucleotide sequences to regulate theexpression of genes for promotor and enhancers; and terminators, all ofwhich are generally used in recombinant DNA technology for producingrecombinant proteins. For example, since a part of and the whole of thenucleotide sequence of SEQ ID NO: 8 function as ribosome-bindingsequences, DNAs to which the part of and the whole of the nucleotidesequence of SEQ ID NO: 8 are ligated upstream of the nucleotidesequences encoding the present non-reducing saccharide-forming enzymecan be arbitrarily used in producing the enzyme as a recombinantprotein.

As described above, the DNAs encoding the present non-reducingsaccharide-forming enzyme should not be restricted to theirorigins/sources, and they are preparable by screening DNAs fromdifferent sources based on hybridization with a DNA comprising anucleotide sequence which encodes at least a part of the amino acidsequence of the enzyme, e.g., the amino acid sequence of SEQ ID NO: 1.Actual examples of these sources are microorganisms of the genusArthrobacter, and preferably, Arthrobacter sp. S34, FERM BP-6450, andits mutants, all of which produce the non-reducing saccharide-formingenzyme. To screen the microorganisms, conventional methods used in thisfield for screening or cloning DNAs such as screening methods ofrecombinant libraries, PCR method, and their modified methods. As aresult of screening, the desired DNAs can be obtained by collecting in ausual manner DNAs confirmed with the expected hybridization. Generally,the DNAs thus obtained comprise a part of or the whole of the nucleotidesequence of SEQ ID NO: 7. For example, a DNA which comprises the wholeof the nucleotide sequence of SEQ ID NO: 7 is generally obtained fromArthrobacter sp. S34, FERM BP-6450. DNAs comprising a part of thenucleotide sequence of SEQ ID NO: 7 can be obtained by similarlyscreening DNAs from microorganisms as sources other than the abovestrain, capable of producing the present non-reducing saccharide-formingenzyme. Such DNAs can be prepared by selecting DNAs, which encode theenzymes having the properties of the present enzyme, from DNAs intowhich have been introduced a replacement, addition and/or deletion ofone or more bases of the above-mentioned DNAs by using one or moreconventional mutation-introducing methods. The DNAs can be also obtainedby applying conventional chemical syntheses based on the nucleotidesequence encoding the present non-reducing saccharide-forming enzyme,e.g., one of SEQ ID NO: 7. Once in hand, the DNAs according to thepresent invention can be easily amplified to the desired level byapplying or using PCR method and autonomously-replicable vectors.

The present DNA encoding the non-reducing saccharide-forming enzymeinclude those in the form of recombinant DNAs which the DNAs have beenintroduced into appropriate vectors. The recombinant DNAs can berelatively-easily preparable by recombinant DNA technology in general ifonly the DNAs are available. Any types of vectors can be used in thepresent invention as long as they autonomously replicable in appropriatehosts. Examples of such vectors are pUC18, pBluescript II SK(+),pKK223-3, λgt•λC, etc., which use Escherichia coli as a host; pUB110,pTZ4, pC194, ρ11, φ1, φ105, etc., which use microorganisms of the genusBacillus; and pHY300PLK, pHV14, TRp7, YEp7, pBS7, etc., which use two ormore microorganisms as hosts. The methods to insert the present DNA intosuch vectors in the present invention may be conventional ones generallyused in this field. A gene containing the present DNA and anautonomously-replicable vector are first digested with a restrictionenzyme and/or ultrasonic disintegrator, then the resultant DNA fragmentsand vector fragments are ligated. The ligation is facilitated by the useof restriction enzymes which specifically act on the cleavage of theDNA, especially, KpnI, AccI, BamHI, BstXI, EcoRI, HindIII, NotI, PstI,SacI, SalI, SmaI, SpeI, XbaI, XhoI, etc. To ligate DNA fragments andvectors, firstly they may be annealed if necessary, then subjected tothe action of a DNA ligase in vivo or in vitro. The recombinant DNA thusobtained can be replicable without substantial limitation in anappropriate host.

The present DNA encoding the non-reducing saccharide-forming enzymefurther includes transformants which the DNA has been introduced intoappropriate vectors. The transformants can be easily preparable byintroducing the DNA or recombinant DNA obtained in the above intoappropriate hosts to transform them. As the hosts, microorganisms andcells from plants and animals, which are used conventionally in thisfield and chosen depending on the vectors in the recombinant DNA, can beused. The microorganisms as hosts include those of the generaEscherichia, Bacillus, and Arthrobacter, and another actinomycetes,yeasts, fungi, etc. To introduce the present DNA into these hostmicroorganisms, conventional competent cell method and protoplast methodcan be used. The present DNA, which encodes the non-reducingsaccharide-forming enzyme introduced into the transformants in thepresent invention, may be present in a separatory form from chromosomesor in an incorporated form into chromosomes. The DNA incorporated intohosts' chromosomes has a character of being stably retained therein andmay be advantageously used in producing the present recombinant protein.

The present non-reducing saccharide-forming enzyme can be obtained in adesired amount by a process for producing the enzyme characterized inthat it comprises the steps of culturing microorganisms capable ofproducing the enzyme, and collecting the produced enzyme from theculture. The microorganisms used in the process can be usedindependently of the genus or the species as long as they produce theenzyme. Examples of such microorganisms are microorganisms of the genusArthrobacter, Arthrobacter sp. S34, FERM BP-6450, and mutants thereof,as well as transformants obtainable by introducing the present DNAencoding the enzyme into appropriate hosts.

Any nutrient culture media used in culturing the process for producingthe present non-reducing saccharide-forming enzyme can be used as longas the aforesaid microorganisms grow therein and produce the enzymewithout restriction to a specific nutrient culture medium. Generally,the nutrient culture media contain carbon and nitrogen sources, and ifnecessary minerals may be added. Examples of the carbon sources aresaccharides such as dextrins, starches, partial starch hydrolysates,glucose, etc., and are saccharide-containing substances such as molassesand yeast extracts, and organic acids such as glucuronic acid andsuccinic acid. The concentration of the carbon sources is chosendepending on the types used, usually 30 w/v %, and preferably 15 w/w %or lower. Examples of the nitrogen sources appropriately used in thepresent invention are inorganic-nitrogen-containing substances such asammonium salts, nitrate, etc.; organic-nitrogen-containing substancessuch as urea, corn steep liquor, casein, peptone, yeast extract, beefextract, etc. Depending on use, it is selectively used among inorganicingredients such as salts of calcium, magnesium, potassium, sodium,phosphoric acid, manganese, zinc, iron, copper, molybdenum, cobalt, etc.

The culture conditions used for producing the present enzyme can be usedselectively from appropriate conditions suitable for growing respectivemicroorganisms used. For example, in the case of using microorganisms ofthe genus Arthrobacter including Arthrobacter sp. S34, FERM BP-6450, thecultivation temperature is usually in the range of 20-50° C., andpreferably 25-37° C.; the cultivation pH is usually in the range of pH4-10, and preferably pH 5-9; and the cultivation time is in the range of10-150 hours. With these conditions, the microorganisms are culturedunder aerobic conditions. When used transformants prepared byintroducing into appropriate hosts the present DNA encoding the presentnon-reducing saccharide-forming enzyme, the transformants are culturedunder aerobic conditions at conditions selected from the cultureconditions such as the culture temperatures of 20-65° C., the culture pHof 2-9, and the culture time of 1-6 days, although they vary dependingon the genus, species, strains or types of microorganisms and vectors.The cultures thus obtained generally contain the present enzyme in cellfractions. In the case of culturing transformants obtained by using ashosts the microorganisms of the genus Bacillus, the resulting culturesmay contain the present enzyme in supernatant fractions depending onvectors used to transform the hosts. The content of the present enzymein the cultures thus obtained is usually 0.01-1,000 units per ml of theculture, though it varies depending on the genus, species or strains ofthe microorganisms and culture conditions used.

The present non-reducing saccharide-forming enzyme is collected from theresulting cultures. The collection method is not restricted; The presentenzyme can be obtained by separating and collecting any one of fractionsof cells and culture supernatants found with a major activity of theenzyme, and if necessary subjecting the collected fraction to anappropriate purification method to collect a purified fractioncontaining the enzyme. To separate the fractions of cells and culturesupernatants of the cultures, conventional solid-liquid separationmethods such as centrifugation and filtration using precoat filters andplain- and hollow fiber-membranes can be arbitrarily used. The desiredfractions are collected from the separated fractions of cells andculture supernatant. For the fraction of cells, the cells are disruptedinto a cell disruptant which is then separated into a cell extract andan insoluble cell fraction, followed by collecting either of the desiredfractions. The insoluble cell fraction can be solubilized byconventional methods, if necessary. As a method to disrupt cells, anyone of techniques of ultrasonication, treatment with cell-wall-lysingenzymes such as lysozyme and glucanase, and load of mechanical press canbe arbitrarily used. To disrupt cells the cultures can be directlytreated with any one of the above techniques, and then resultingmixtures are treated with any one of the above solid-liquid separationmethods to collect a liquid fraction. Thus a cell extract can bearbitrarily obtained.

The methods used for more purifying the present non-reducingsaccharide-forming enzyme include conventional ones to purifysaccharide-related enzymes in general such as salting out, dialysis,filtration, concentration, gel filtration chromatography, ion-exchangechromatography, hydrophobic chromatography, reverse-phasechromatography, affinity chromatography, gel electrophoresis and,isoelectric point electrophoresis. These methods can be used incombination depending on purposes. From the resulting fractionsseparated by these methods, fractions with a desired activity assayed bythe method for non-reducing saccharide-forming enzyme are collected toobtain the present non-reducing saccharide-forming enzyme purified to adesired level. According to the methods in the later described Examples,the present enzyme can be purified up to an electrophoreticallyhomogenous level. As described above, the present method provide thepresent non-reducing saccharide-forming enzyme in the form of a culture,cell fraction, fraction of culture supernatant, cell disruptant, cellextract, soluble and insoluble cell-fraction, partially purified enzymefraction, and purified enzyme fraction. These fractions may containanother type of the present trehalose-releasing enzyme. The non-reducingsaccharide-forming enzyme thus obtained can be immobilized in a usualmanner before use. The methods for immobilization are, for example,binding method to ion exchangers, covalent bonding/adsorption to and onresins and membranes, and entrapping immobilization method using highmolecular weight substances. The non-reducing saccharide-forming enzymethus obtained can be arbitrarily used in processes for producingsaccharides including the later described present process for producingsaccharide. Particularly, since the present non-reducingsaccharide-forming enzyme has an optimum temperature in a mediumtemperature range and preferably has an optimum pH in an acid pH range,it can be advantageously used to produce saccharides when used incombination with the later described present trehalose-releasing enzyme,starch-debranching enzyme having an optimum pH in an acid pH range, andcyclomaltodextrin glucanotransferase that effectively acts at mediumtemperature range.

Explaining the present trehalose-releasing enzyme based on the aminoacid sequence, the enzyme has the amino acid sequence of SEQ ID NO: 9 asa whole, and has the amino acid sequences of SEQ ID NOs: 10 to 16 aspartial amino acid sequences in some cases. In addition to these enzymeshaving the whole of the above-identified amino acid sequences, thepresent invention includes another types of enzymes which comprise apart of any one of the amino acid sequences selected therefrom or whichhave both the action as the present trehalose-releasing enzyme and theabove-identified optimum temperature. Examples of the amino acidsequences of such enzymes are those which contain, within the amino acidsequences, a partial amino acid sequence or an amino acid residue whichrelate to the expression of the properties of the present non-reducingsaccharide-forming enzyme, and which one or more amino acids arereplaced with different amino acids, added thereunto and/or deletedtherefrom other than the above partial amino acid sequence or the aminoacid residue. Examples of amino acid sequences replaced with differentamino acids as referred to in the present invention include those whichless than 30% and preferably less than 20% of the amino acid sequencescomposing the amino acid sequence of SEQ ID NO: 9 are replaced withanother amino acids which have similar properties and structures torespective ones to be replaced. Examples of groups of such amino acidsare a group of aspartic acid and glutamic acid as acid amino acids, oneof lysine, arginine, and histidine as basic amino acids, one ofasparagine and glutamine as amid-type amino acids, one of serine andthreonine as hydroxyamino acids, and one of valine, leucine andisoleucine as branched-chain amino acids. Examples of another amino acidsequences of the enzyme containing a part of any one of the amino acidsequences selected from SEQ ID NOs: 9 to 16 are those which might have asubstantially similar stereo-structure to the one of the amino acidsequence of SEQ ID NO: 9, i.e., replacement, deletion and/or addition ofamino acid(s) are introduced into the amino acid sequence of SEQ ID NO:9. The stereo-structure of proteins is estimable by screeningcommercially available databases for stereo-structures of proteins whichhave amino acid sequences related to the aiming ones and have revealedstereo-structures, referencing the screened stereo-structures, and usingcommercially available soft wares for visualizing stereo-structures. Theabove-identified amino acid sequence of the present trehalose-releasingenzyme has a homology of at least 60%, preferably at least 70%, and morepreferably at least 80% to SEQ ID NO: 9.

As described above, the trehalose-releasing enzyme should not berestricted to a specific origin/source. Examples of such are thosederived from microorganisms, i.e., those of the genus Arthrobacter,Arthrobacter sp. S34, FERM BP-6450, and mutants thereof. The mutants canbe obtained by treating in a usual manner Arthrobacter sp. S34, FERMBP-6450, with known mutagens such asN-methyl-N′-nitro-N-nitrosoguanidine, ethyl methanesulfonate,ultraviolet, and transposon; screening the desired mutants capable ofproducing a non-reducing saccharide-forming enzyme and having an optimumtemperature at temperatures in a medium temperature range, and usuallyat temperatures in the range of over 45° C. but below 60° C. The enzymefrom Arthrobacter sp. S34, FERM BP-6450, usually has the amino acidsequences of SEQ ID NOs: 9 to 16. Another non-reducingsaccharide-forming enzymes from microorganisms of mutants Arthrobactersp. S34, FERM BP-6450, and another microorganisms comprise the whole ora part of any one of the amino acid sequences of SEQ ID NOs: 9 to 16.Concrete examples of another enzymes include recombinant enzymes whichact as the present trehalose-releasing enzyme and have an optimumtemperature at temperatures in a medium temperature range, and usuallyat temperatures of over 45° C. but below 60° C. The recombinant enzymescan be obtainable by applying the recombinant DNA technology for the DNAencoding the present trehalose-releasing enzyme, and have the whole or apart of any one of the amino acid sequences of SEQ ID NOs: 9 to 16.

Most of the trehalose-releasing enzyme according to the presentinvention has the following physicochemical properties:

-   -   (1) Action Specifically hydrolyses a non-reducing saccharide        having a trehalose structure as an end unit to release trehalose        from the rest of the non-reducing saccharide;    -   (2) Molecular weight About 62,000±5,000 daltons on sodium        dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE);    -   (3) Isoelectric point (PI) About 4.7±0.5 on isoelectrophoresis        using ampholyte;    -   (4) Optimum temperature About 50° C. to about 55° C. when        incubated at pH 6.0 for 30 min;    -   (5) Optimum pH About 6.0 when incubated at 50° C. for 30 min;    -   (6) Thermal stability Stable up to a temperature of about 50° C.        when incubated at pH 7.0 for 60 min; and    -   (7) pH Stability Stable at pHs of about 4.5 to about 10.0 when        incubated at 4° C. for 24 hours.

The present trehalose-releasing enzyme can be obtained in a prescribedamount by the later described present process for producing the same.

The present invention provides a DNA encoding the presenttrehalose-releasing enzyme. Such a DNA is quite useful in producing theenzyme in the form of a recombinant protein. In general, the DNAincludes those which encode the enzyme independently of itsorigin/source. Examples of such a DNA are those which contain the wholeor a part of the nucleotide sequence of SEQ ID NO: 17 or complementaryones thereunto. The DNA comprising the whole of the nucleotide sequenceof SEQ ID NO: 17 encodes the amino acid sequence of SEQ ID NO: 9. TheDNAs, which contain the whole or a part of the nucleotide sequence ofSEQ ID NO: 17, include those which have a nucleotide sequencecorresponding to an amino acid sequence relating to the expression ofthe properties of the present non-reducing saccharide-forming enzyme,and have the nucleotide sequence of SEQ ID NO: 17 introduced with areplacement, deletion and/or addition of one or more bases whileretaining the nucleotide sequence relating to the expression of theproperties of the present trehalose-releasing enzyme. The DNAs accordingto the present invention should include those which one or more basesare replaced with different ones based on the degeneracy of geneticcode. Also the DNAs according to the present invention include thosewhich comprise the nucleotide sequences that encode the presenttrehalose-releasing enzyme and further comprise additional one or moreanother nucleotide sequences selected from the group consisting ofribosome-binding sequences such as an initiation codon, terminationcodon, and Shine-Dalgarno sequence; nucleotide sequences encoding signalpeptides, recognition sequences for appropriate restriction enzymes;nucleotide sequences to regulate the expression of genes for promotorand enhancers; and terminators, all of which are generally used inrecombinant DNA technology for producing recombinant proteins. Forexample, since a part of and the whole of the nucleotide sequence of SEQID NO: 8 function as ribosome-binding sequences, DNAs to which the partof and the whole of the nucleotide sequence of SEQ ID NO: 8 are ligatedupstream of the nucleotide sequences encoding the presenttrehalose-releasing enzyme can be arbitrarily used in producing theenzyme as a recombinant protein.

As described above, the DNAs encoding the present trehalose-releasingenzyme should not be restricted to their origins/sources, and they arepreparable by screening DNAs from different sources based onhybridization with a DNA comprising a nucleotide sequence which encodesat least a part of the amino acid sequence of the enzyme, e.g., theamino acid sequence of SEQ ID NO: 9. Actual examples of these sourcesare microorganisms of the genus Arthrobacter, and preferably,Arthrobacter sp. S34, FERM BP-6450, and its mutants, all of whichproduce the non-reducing saccharide-forming enzyme. To screen themicroorganisms, conventional methods used in this field for screening orcloning DNAs such as screening methods of recombinant libraries, PCRmethod, and their modified methods. As a result of screening, thedesired DNAs can be obtained by collecting in a usual manner DNAsconfirmed with the expected hybridization. Generally, the DNAs thusobtained comprise a part of or the whole of the nucleotide sequence ofSEQ ID NO: 17. For example, a DNA which comprises the whole of thenucleotide sequence of SEQ ID NO: 17 is generally obtained fromArthrobacter sp. S34, FERM BP-6450. DNAs comprising a part of thenucleotide sequence of SEQ ID NO: 17 can be obtained by similarlyscreening DNAs from microorganisms as sources other than the abovestrain, capable of producing the trehalose-releasing enzyme. Such DNAscan be prepared by selecting DNAs, which encode the enzymes having theproperties of the enzyme, from DNAs into which have been introduced areplacement, addition and/or deletion of one or more bases of theabove-mentioned DNAs by using one or more conventionalmutation-introducing methods. The DNAs can be also obtained by applyingconventional chemical syntheses based on the nucleotide sequenceencoding the present trehalose-releasing enzyme, e.g., one of SEQ ID NO:17. Once in hand, the DNAs according to the present invention can beeasily amplified to the desired level by applying or using PCR methodand autonomously-replicable vectors.

The present DNA encoding the trehalose-releasing enzyme include those inthe form of recombinant DNAs which the DNAs have been introduced intoappropriate vectors. The recombinant DNAs can be relatively-easilypreparable by recombinant DNA technology in general if only the DNAs areavailable. Any types of vectors can be used in the present invention aslong as they autonomously replicable in appropriate hosts. Examples ofsuch vectors are pUC18, pBluescript II SK(+), pKK223-3, λgt•λC, etc.,which use Escherichia coli as a host; pUB110, pTZ4, pC194, ρ11, φ1,φ105, etc., which use microorganisms of the genus Bacillus; andpHY300PLK, pHV14, TRp7, YEp7, pBS7, etc., which use two or moremicroorganisms as hosts. The methods to insert the present DNA into suchvectors in the present invention may be conventional ones generally usedin this field. A gene containing the present DNA and anautonomously-replicable vector are first digested with a restrictionenzyme and/or ultrasonic disintegrator, then the resultant DNA fragmentsand vector fragments are ligated. The ligation is facilitated by the useof restriction enzymes which specifically act on the cleavage of theDNA, especially, KpnI, AccI, BamHI, BstXI, EcoRI, HindIII, NotI, PstI,SacI, SalI, SmaI, SpeI, XbaI, XhoI, etc. To ligate DNA fragments andvectors, firstly they may be annealed if necessary, then subjected tothe action of a DNA ligase in vivo or in vitro. The recombinant DNA thusobtained can be replicable without substantial limitation in anappropriate host.

The present DNA encoding the trehalose-releasing enzyme further includestransformants which the DNA has been introduced into appropriatevectors. The transformants can be easily preparable by introducing theDNA or recombinant DNA obtained in the above into appropriate hosts totransform them. As the hosts, microorganisms and cells from plants andanimals, which are used conventionally in this field and chosendepending on the vectors in the recombinant DNA, can be used. Themicroorganisms as hosts include those of the genera Escherichia,Bacillus, and Arthrobacter, and another actinomycetes, yeasts, fungi,etc. To introduce the present DNA into these host microorganisms,conventional competent cell method and protoplast method can be used.The present DNA, which encodes the trehalose-releasing enzyme introducedinto the transformants in the present invention, may be present in aseparatory form from chromosomes or in an incorporated form intochromosomes. The DNA incorporated into hosts' chromosomes has acharacter of being stably retained therein and may be advantageouslyused in producing the present recombinant protein.

The aforesaid techniques used for obtaining the present DNAs includingrecombinant DNAs and transformants, and the techniques for obtaining theDNAs and recombinant proteins are commonly used in the art; For example,J. Sumbruck et al. in “Molecular Cloning A Laboratory Manual”, 2ndedition, published by Cold Spring Harbor Laboratory Press (1989),discloses in detail methods for obtaining desired DNAs and applicationsfor production use of the obtained DNAs. For example, Japanese PatentNo. 2,576,970 discloses a method for stabilizing a transformed DNA,which uses as a host a microorganism defective in an aiming gene.Japanese Patent Kokai No. 157,987/88 discloses a vector whicheffectively expresses an aiming DNA in microorganisms of the genusBacillus. Japanese Patent Kohyo No. 502,162/93 discloses a method forstably introducing a desired DNA into a bacterial chromosome. JapanesePatent Kohyo No. 506,731/96 discloses an efficient production method ofa starch hydrolysing enzyme, using recombinant DNA technology. JapanesePatent Kohyo Nos. 500,543/97 and 500,024/98 disclose a host-vectorsystem using fungi for efficient production of recombinant proteins.These methods conventionally used in the art are arbitrarily applicablefor the present invention.

In the art, when the desired DNAs are available by the above methods,there have been commonly provided transformants which the DNAs areintroduced into appropriate plants and animals, i.e., transgenic plantsand animals. The present DNA, which encodes the non-reducingsaccharide-forming enzyme and the trehalose-releasing enzyme in the formof a DNA introduced into appropriate hosts, also includes the transgenicplants and animals. To obtain the transgenic animals, it is obtained asa whole by a process comprising the DNA which encodes either of thepresent enzymes alone or together with other desired DNA such as apromotor and enhancer into an appropriate vector selected depending onthe species of the host animal, introducing the resulting recombinantDNA into a fertilized egg or embryonic stem cell from the host animal bya method such as micro-injection and electroporation, or by an infectionmethod using recombinant viruses containing the recombinant DNA.Examples of the host animals are conventional experimental rodents suchas mice, rats, and hamsters; and mammals conventionally used as domesticanimals such as goats, sheep, pigs, and cows, all of which have anadvantage of being bred easily. The resulting cells introduced with theDNA are transplanted in uterine tube or uterus of a pseudopregnancyfemale animal of the same species as the cells. Thereafter, transgenicanimals, which have been introduced with the DNA encoding the presentenzymes by applying hybridization or PCR method, are obtained fromnewborns in a natural or cesarean sectional manner. Thus the present DNAin the form of a transgenic animal can be obtained. Referring totransgenic animals, they are disclosed in detail in“Jikken-Igaku-Bessatsu-Shin-Idennshi-Kogaku-Handbook” (Handbook ofGenetic Engineering), pp. 269-283 (1996), edited by Masami MATSUMURA,Hiroto OKAYAMA, and Tadashi YAMAMOTO, published by Yodosha Co., Ltd.,Tokyo, Japan. The method for obtaining transgenic plants comprises, forexample, providing a plasmid as a vector of a microorganism of the genusAgrobacterium infectious to plants, introducing the DNA encoding eitherof the present enzymes into the vector, and either introducing theresulting recombinant DNA into plant bodies or protoplasts, or coatingheavy metal particles with a DNA including nucleotide sequence encodingeither of the present enzymes and directly injecting the coatedparticles into plant bodies or protoplasts using a particle gun.Although various types of plants can be used as host plants, theygenerally include edible plants such as potato, soybean, wheat, burley,rice, corn, tomato, lettuce, alfalfa, apple, peach, melon, etc. Byapplying hybridization or PCR method for the above transformed plantbodies and protoplasts, transformants containing the desired DNA areselected. The transformed protoplasts can be regenerated into plantbodies as the present DNA in the form of transgenic plants. Thetechniques of transgenic plants are generally disclosed in GeneticEngineering, edited by Jane K. Setlow, published by Plenum PublishingCorporation, NY, USA, Vol. 16, pp. 93-113 (1994). The DNA in the form ofthe aforesaid transgenic animals and plants can be used as sources ofthe present non-reducing saccharide-forming enzyme and/ortrehalose-releasing enzyme, and used as edible plants and animals whichcontain trehalose or non-reducing saccharide having a trehalosestructure.

The present trehalose-releasing enzyme can be obtained in a desiredamount by the present process for producing the enzyme which ischaracterized in that it comprises culturing a microorganism capable ofproducing the enzyme in a nutrient culture medium, and collecting theproduced enzyme from the resulting culture. Any microorganisms can beused in the present process independently of their genus and species aslong as they produce the present trehalose-releasing enzyme. Examples ofsuch microorganisms are those of the genus Arthrobacter, Arthrobactersp. S34, FERM BP-6450, and mutants thereof, as well as transformantsobtainable by introducing the present DNA encoding the enzyme intoappropriate host microorganisms.

Any nutrient culture media for culturing the process for producing thepresent trehalose-releasing enzyme can be used as long as the aforesaidmicroorganisms grow therein and produce the enzyme without restrictionto a specific nutrient culture medium. Generally, the nutrient culturemedia contain carbon and nitrogen sources, and if necessary minerals maybe added. Examples of the carbon sources are saccharides such asdextrins, starches, partial starch hydrolysates, glucose, etc., and aresaccharide-containing substances such as molasses and yeast extracts,and organic acids such as glucuronic acid and succinic acid. Theconcentration of the carbon sources is chosen depending on the typesused, usually 30 w/v %, and preferably 15 w/w % or lower. Examples ofthe nitrogen sources appropriately used in the present invention areinorganic-nitrogen-containing substances such as ammonium salts,nitrate, etc.; organic-nitrogen-containing substances such as urea, cornsteep liquor, casein, peptone, yeast extract, beef extract, etc.Depending on use, it is selectively used among inorganic ingredientssuch as salts of calcium, magnesium, potassium, sodium, phosphoric acid,manganese, zinc, iron, copper, molybdenum, cobalt, etc.

The culture conditions used for producing the presenttrehalose-releasing enzyme can be used selectively from appropriateconditions suitable for growing respective microorganisms used. Forexample, in the case of using microorganisms of the genus Arthrobacterincluding Arthrobacter sp. S34, FERM BP-6450, the cultivationtemperature is usually in the range of 20-50° C., and preferably 25-37°C.; the cultivation pH is usually in the range of pH 4-10, andpreferably pH 5-9; and the cultivation time is in the range of 10-150hours. With these conditions, the microorganisms are cultured underaerobic conditions. When used transformants prepared by introducing intoappropriate hosts the present DNA encoding the trehalose-releasingenzyme, the transformants are cultured under aerobic conditions atconditions selected from the culture conditions such as the culturetemperatures of 20-65° C., the culture pH of 2-9, and the culture timeof 1-6 days, although they vary depending on the genus, species, strainsor types of microorganisms and vectors. The cultures thus obtainedgenerally contain the enzyme in cell fractions. In the case of culturingtransformants obtained by using as hosts the microorganisms of the genusBacillus, the resulting cultures may contain the enzyme in supernatantfractions depending on vectors used to transform the hosts. The contentof the enzyme in the cultures thus obtained is usually 0.01-3,000 unitsper ml of the culture, though it varies depending on the genus, speciesor strains of the microorganisms and culture conditions used.

The present trehalose-releasing enzyme is collected from the resultingcultures. The collection method is not restricted; The enzyme can beobtained by separating and collecting any one of fractions of cells andculture supernatants found with a major activity of the enzyme, and ifnecessary subjecting the collected fraction to an appropriatepurification method to collect a purified fraction containing theenzyme. To separate the fractions of cells and culture supernatants ofthe cultures, conventional solid-liquid separation methods such ascentrifugation and filtration using precoat filters and plain- andhollow fiber-membranes can be arbitrarily used. The desired fractionsare collected from the separated fractions of cells and culturesupernatant. For the fraction of cells, the cells are disrupted into acell disruptant which is then separated into a cell extract and aninsoluble cell fraction, followed by collecting either of the desiredfractions. The insoluble cell fraction can be solubilized byconventional methods, if necessary. As a method to disrupt cells, anyone of techniques of ultrasonication, treatment with cell-wall-lysingenzymes such as lysozyme and glucanase, and load of mechanical press canbe arbitrarily used. To disrupt cells the cultures can be directlytreated with any one of the above techniques, and then resultingmixtures are treated with any one of the above solid-liquid separationmethods to collect a liquid fraction. Thus a cell extract can bearbitrarily obtained.

The methods used for more purifying the present trehalose-releasingenzyme include conventional ones to purify saccharide-related enzymes ingeneral such as salting out, dialysis, filtration, concentration, gelfiltration chromatography, ion-exchange chromatography, hydrophobicchromatography, reverse-phase chromatography, affinity chromatography,gel electrophoresis and, isoelectric point electrophoresis. Thesemethods can be used in combination depending on purposes. From theresulting fractions separated by these methods, fractions with a desiredactivity assayed by the method for trehalose-releasing enzyme arecollected to obtain the enzyme purified to a desired level. According tothe methods in the later described Examples, the present enzyme can bepurified up to an electrophoretically homogenous level. As describedabove, the present method provide the present trehalose-releasing enzymein the form of a culture, cell fraction, fraction of culturesupernatant, cell disruptant, cell extract, soluble and insolublecell-fraction, partially purified enzyme fraction, and purified enzymefraction. These fractions may contain another type of the presentnon-reducing saccharide-forming enzyme. The present trehalose-releasingenzyme thus obtained can be immobilized in a usual manner before use.The methods for immobilization are, for example, binding method to ionexchangers, covalent bonding/adsorption to and on resins and membranes,and entrapping immobilization method using high molecular weightsubstances. The trehalose-releasing enzyme thus obtained can bearbitrarily used in processes for producing saccharides including thelater described present process for producing saccharide. Particularly,since the trehalose-releasing enzyme has an optimum temperature in amedium temperature range and preferably has an optimum pH in an acid pHrange, it can be advantageously used to produce saccharides when used incombination with the later described present trehalose-releasing enzyme,starch-debranching enzyme having an optimum pH in an acid pH range, andcyclomatodextrin glucanotransferase that effectively acts attemperatures in a medium temperature range.

The present invention provides a process for producing saccharidescomprising non-reducing saccharides by using the aforesaid presentenzymes; the process comprising the steps of allowing the non-reducingsaccharide-forming enzyme and/or the trehalose-releasing enzyme to acton reducing partial starch hydrolysates to form non-reducingsaccharides, and collecting the resulting non-reducing saccharides orsaccharide compositions with a lesser reducibility. In the process, theuse of one or more another types of non-reducing saccharide-formingenzymes and trehalose-releasing enzymes other than the present enzymes,and other saccharide-related enzymes should not be excluded from thepresent invention. The reducing partial starch hydrolysates used in theprocess can be used independently of their origins/sources. Thenon-reducing saccharides as referred to in the present invention includenon-reducing saccharides in general such as trehalose and those having atrehalose structure.

The reducing partial starch hydrolysates used in the present process forproducing saccharides can be obtained, for example, by liquefyingstarches or amylaceous substances by conventional methods. The starchesinclude terrestrial starches such as corn starch, rice starch, and wheatstarch; and subterranean starches such as potato starch, sweet potatostarch, and tapioca starch. To liquefy these starches, they aregenerally suspended in water into starch suspensions, preferably, thosewith a concentration of at least 10 w/w %, and more preferably thosewith a concentration of about 20 to about 50 w/w %, and treated withmechanical, acid and/or enzymatic treatments. Relatively-lower degree ofliquefaction is satisfactorily used, preferably, DE (dextroseequivalent) of less than 15, and more preferably DE of less than 10.When liquefied with acids, the starches are treated with hydrochloricacid, phosphoric acid, oxalic acid, etc., and then the resultingmixtures are neutralized with calcium carbonate, calcium oxide, sodiumcarbonate, etc., to desired pHs before use. To liquefy the starches withenzymes, α-amylase, particularly, and thermostable liquefying α-amylaseare satisfactorily used. The liquefied starches thus obtained can befurther subjected to the action of α-amylase, maltotriose-formingamylase, maltotetraose-forming amylase, maltopentaose-forming amylase,maltohexaose-forming amylase, etc., and the resulting reaction mixturescan be used as the reducing partial starch hydrolysates. The propertiesof the starch-related enzymes are described in detail in Handbook ofAmylases and Related Enzymes, pp. 18-81, and pp. 125-142 (1988),published by Pergamon Press.

The reducing partial starch hydrolysates thus obtained are subjected tothe action of the present non-reducing saccharide-forming enzyme and/ortrehalose-releasing enzyme, and if necessary further subjected to theaction of one or more starch-related enzymes such as α-amylase,β-amylase, glucoamylase, starch debranching enzymes such as isoamylaseand pullulanase, cyclomaltodextrin glucanotransferase, α-glucosidase,and β-fructofuranosidase. Conditions used for enzymatic reactions arethose suitable for enzymes used; Usually they are selected from pHs 4-10and temperatures of 20-70° C. and preferably pHs 5-7 and temperatures of30-60° C. Particularly, non-reducing saccharides can be effectivelyproduced by enzymatic reactions at temperatures in a medium temperaturerange, i.e., temperatures of over 40° C. but below 60° C. or over 45° C.but below 60° C., and pHs of slight acid or acid pH conditions. Theorder of allowing the enzymes to act on reducing partial starchhydrolysates is not restricted; one proceeds or follows another one, orplural enzymes can be arbitrarily allowed to act on substratessimultaneously.

The amount of enzymes is appropriately set depending on enzymaticconditions and reaction times, and final uses of non-reducingsaccharides or less-reducible saccharide compositions containingthereof. For the present non-reducing saccharide-forming enzyme andtrehalose-releasing enzyme, the former is used in an amount of about0.01 to about 100 units/g solid of reducing partial starch hydrolysates,and the latter is used in an amount of about 1 to about 10,000 units/gsolid of reducing partial starch hydrolysates. Cyclomatodextringlucanotransferase is used in an amount of about 0.05 to about 500units/g reducing partial starch hydrolysates, d.s.b. The reactionmixtures obtained with these enzymes usually contain trehalose,α-glucosyltrehalose, α-maltosyltrehalose, α-maltotriosyltrehalose,α-maltotetraosyltrehalose, or α-maltopentaosyltrehalose. In the aboveprocess, when used in combination, the present non-reducingsaccharide-forming enzyme and trehalose-releasing enzyme along with astarch debranching enzyme and cyclomatodextrin glucanotransferasecharacteristically more produce a large amount of trehalose and arelatively-lower molecular weight of non-reducing saccharide having atrehalose structure.

From the resulting reaction mixtures, non-reducing saccharides andsaccharide compositions with a lesser reducibility are collected. Inthese production steps, conventionally used processed for saccharidescan be appropriately selected. The resulting reaction mixtures aresubjected to filtration and centrifugation to remove insolublesubstances, and then the resultant solutions are purified bydecoloration with an activated charcoal, desalted with ion exchangers inH- and OH-form, and concentrated into syrupy products. If necessary, thesyrupy products can be further purified into non-reducing saccharideswith a relatively-high purity; In the purification, one or more methods,for example, column chromatographic fractionations such as ion-exchangecolumn chromatography, column chromatography using an activated charcoalor a silica gel; separatory sedimentation using organic acids such asacetone and alcohol; separation using membranes with an appropriateseparability; and alkaline treatments to decompose and remove theremaining reducing saccharides. In particular, ion-exchange columnchromatography can be suitably used in the present invention as anindustrial-scale preparation of the object saccharides. Non-reducingsaccharides with an improved purity can be arbitrary prepared by, forexample, column chromatography using a strongly-acid cation exchangeresin as described in Japanese Patent Kokai Nos. 23,799/83 and 72,598/83to remove concomitant saccharides. In this case, any of fixed-bed,moving bed, and semi-moving methods can be employed.

If necessary, the resulting non-reducing saccharides or a relatively-lowreducing saccharides containing the non-reducing saccharides can behydrolyzed by amylases such as α-amylase, β-amylase, glucoamylase andα-glucosidase to control their sweetness and reducing power or to lowertheir viscosity; and the products thus obtained can be further treatedwith processings where the remaining reducing saccharides arehydrogenated into sugar alcohols to diminish their reducing powder.Particularly, trehalose can be easily prepared by allowing glucoamylaseor α-glucosidase to act on the non-reducing saccharides orrelatively-low reducing saccharides containing the non-reducingsaccharides. A high trehalose content fraction is obtainable by allowingglucoamylase or α-glucosidase to act on these saccharides to form amixture of trehalose and glucose, and subjecting the mixture to theaforesaid purification methods such as column chromatography using ionexchangers to remove glucose. The high trehalose content fraction can bearbitrary purified and concentrated into a syrupy product. If necessary,the syrupy product can be concentrated into a supersaturated solution,followed by crystallizing hydrous or anhydrous crystalline trehalose andrecovering the resultant crystal.

To produce hydrous crystalline trehalose, an about 65-90 w/w % solutionof trehalose with a purity of about 60 w/w % or higher is placed in acrystallizer, and if necessary in the presence of 0.1-20 w/v % seedcrystal, gradually cooled while stirring at a temperature of 95° C. orlower, and preferably at a temperature of 10-90° C. to obtain amassecuite containing hydrous crystalline trehalose. Continuouscrystallization method to effect crystallization under concentratingconditions in vacuo can be arbitrarily used.

Conventional methods such as separation, block pulverization,fluidized-bed granulation, and spray drying can be employed in theinvention to prepare from the massecuite hydrous crystalline trehaloseor crystalline saccharides containing the trehalose crystal.

In the case of separation, massecuites are usually subjected to abasket-type centrifuge to separate hydrous crystalline trehalose from amother liquor, and if necessary the hydrous crystalline trehalose iswashed by spraying with a small amount of cold water to facilitate thepreparation of hydrous crystalline trehalose with a higher purity. Inthe case of spray drying, crystalline saccharides with no orsubstantially free of hygroscopicity are easily prepared by sprayingmassecuites with a concentration of 70-85 w/w %, on a dry solid basis(d.s.b.), and a crystallinity of about 20-60%, d.s.b., from a nozzle bya high-pressure pump; drying the resultant products with air heated to60-100° C. which does not melt the resultant crystalline powders; andaging the resultant powders for about 1 to about 20 hours while blowingthereto air heated to 30-60° C. In the case of block pulverization,crystalline saccharides with no or substantially free of hygroscopicityare easily prepared by allowing massecuites with a moisture content of10-20 w/w % and a crystallinity of about 10-60%, d.s.b., to stand forabout 0.1 to about 3 days to crystallize and solidify the whole contentsinto blocks; and pulverizing or cutting the resultant blocks.

To produce anhydrous crystalline trehalose, the hydrous crystallinetrehalose obtained in the above is dried at a normal or reduced pressureat temperatures of 70-160° C., and preferably at 80-100° C.; or arelatively-high concentration and content trehalose solution with amoisture content of less than 10% is placed in a crystallizer, stirredin the presence of a seed crystal at temperatures of 50-160° C., andpreferably 80-140° C. to produce a massecuite containing anhydrouscrystalline trehalose, and treating the massecuite with methods such asblock pulverization, fluidized-bed granulation, and spray drying underrelatively-high temperatures and drying conditions.

The non-reducing saccharides or saccharide composition, containingthereof with a relatively-low reducibility, thus obtained are low inreducibility and satisfactory in stability; they do not become browning,form indisagreeable smell, and deteriorate the following anothermaterials when mixed and processed with another materials, for example,amino-acid-containing substances such as amino acids, oligopeptides, andproteins. Even with a relatively-low reducibility, the above-identifiedsaccharides have a relatively-low viscosity, and those with arelatively-low average glucose polymerization degree have arelatively-high quality and sweetness. These saccharides can bearbitrarily used in the fields of foods, cosmetics, and pharmaceuticals,etc., as disclosed in Japanese Patent Kokai Nos. 66,187/96, 66,188/96,73,482/96, 73,506/96, 73,504/96, 336,363/96, 9,986/97, 154,493/97,252,719/97, 66,540/98, and 168,093/98; and Japanese Patent ApplicationNos. 236,441/97, 256,219/97, 268,202/97, 274,962/97, 320,519/97,338,294/97, 55,710/98, 67,628/98, 134,553/98 and 214,375/98, which wereall applied for by the same applicant as the present applicant.

The following examples describe the present invention in more detail:

EXAMPLE 1

Microorganism Capable of Producing Non-Reducing Saccharide-FormingEnzyme and Trehalose-Releasing Enzyme

The present inventors widely screened soils to isolate a microorganismcapable of producing non-reducing saccharide-forming enzyme andtrehalose-releasing enzyme. As a result, they isolated a microorganismwith such a property from a soil in Ako, Hyogo, Japan, and identifiedthe microorganisms in accordance with the method as described in“Biseibutsu-no-Bunrui-to-Dotei” (Classification and Identification ofMicroorganisms), edited by Takeji Hasegawa, published by JapanScientific Societies Press, Tokyo, Japan (1985). The results were asfollows:

-   -   Results on Cell Morphology    -   (1) Characteristics of cells when incubated at 37° C. in        nutrient agar broth Usually existing a rod form of        0.4-0.5×0.8-1.2 μm; Existing in a single form but uncommonly        existing in a polymorphic form;        -   Free of motility;        -   Asporogenic;        -   Non-acid fast; and        -   Gram stain: Positive.    -   (2) Characteristics of cells when incubated at 37° C. in EYG        nutrient agar        -   Exhibiting a growth cycle of rods and cocci.    -   Results on Cultural Property        -   (1) Characteristics of colony formed when incubated at            37° C. in nutrient agar broth plate            -   Shape: Circular colony having a diameter of about 1-2 mm                after 2-days incubation;            -   Rim: Entire;            -   Projection: Convex;            -   Gloss: Moistened gloss;            -   Surface: Plain; and            -   Color: Semi-transparent or cream.        -   (2) Characteristics of colony formed when incubated at            37° C. in nutrient agar broth slant            -   Growth: Satisfactory; and            -   Shape: Thread-like.        -   (3) Characteristics of colony formed when incubated at            37° C. in agar slant with yeast extract and peptone            -   Growth: Satisfactory; and            -   Shape: Thread-like.        -   (4) Characteristics of colony formed when stab-cultured at            27° C. in nutrient gelatin broth Not liquefying gelatin.    -   Results on Physiological Properties        -   (1) Methyl red test: Negative        -   (2) VP-test: Positive        -   (3) Formation of indole: Negative        -   (4) Formation of hydrogen sulfide: Negative        -   (5) Hydrolysis of starch: Positive        -   (6) Liquefaction of gelatin: Negative        -   (7) Utilization of citric acid: Positive        -   (8) Utilization of inorganic nitrogen source:            -   Utilizing nitrate but not ammonium salts        -   (9) Formation of pigment: Non        -   (10) Urease: Negative        -   (11) Oxidase: Negative        -   (12) Catalase: Positive        -   (13) Growth range: Growing at pHs of 4.5-8.0 and            temperatures of 20-50° C.; and            -   Optimum temperatures of 30-45° C.        -   (14) Oxygen requirements: Aerobic        -   (15) Utilization of carbon sources            -   L-Arabinose: Assimilated            -   D-Glucose: Assimilated            -   D-Fructose: Not assimilated            -   D-Galactose: Not assimilated            -   L-Rhamnose: Not assimilated            -   D-Xylose: Not assimilated            -   D-Mannose: Assimilated            -   Raffinose: Not assimilated            -   Trehalose: Not assimilated            -   Sucrose: Not assimilated            -   Maltose: Not assimilated            -   Lactose: Not assimilated            -   D-Dulcitol: Not assimilated            -   D-Mannitol: Not assimilated            -   Gluconic acid: Assimilated            -   Succinic acid: Assimilated            -   Nicotinic acid: Not assimilated            -   L-Maleic acid: Assimilated            -   Acetic acid: Assimilated            -   Lactic acid: Assimilated        -   (16) Acid formation from sugars            -   L-Arabinose: Slightly formed            -   D-Glucose: Slightly formed            -   D-fructose: Not formed            -   D-Galactose Slightly formed            -   L-Rhamnose Slightly formed            -   D-Xylose: Slightly formed            -   Glycerol: Slightly formed            -   Raffinose: Not formed            -   Trehalose: Slightly formed            -   Sucrose: Slightly formed            -   Maltose: Slightly formed            -   Lactose: Not formed        -   (17) Utilization of amino acid Not utilizing sodium            L-glutamate, sodium L-aspartate, L-histidine and L-arginine,        -   (18) Decarboxylase test on amino acid Negative against            L-lysine, L-ornithine and L-arginine.        -   (19) DNase: Negative        -   (20) N-Acyl type of cell wall: Acetyl        -   (21) Main diamino acid of cell wall: Lysine        -   (22) Mol % of guanine (G) plus cytosine (C) of DNA: 71.2%

These bacteriological properties were compared with those of knownmicroorganisms with reference to Bergey's Manual of SystematicBacteriology, Vol. 2 (1984). As a result, it was revealed that themicroorganism was identified as a novel one of the genus Arthrobacter.Based on the results, the present inventors named this microorganism“Arthrobacter sp. S34”. The microorganisms was deposited and accepted onAug. 6, 1998, under the accession number of FERM BP-6450 in and by thePatent Microorganism Depository, National Institute of Bioscience andHuman-Technology Agency of Industrial Science & Technology, Ministry ofInternational Trade & industry, 1-3, Higashi, 1 chome, Tsukuba-shi,Ibaraki-ken 305-8566, Japan.

The homology of DNA between the identified microorganism andtype-strains of the genus Arthrobacter, deposited in American TypeCulture Collection (ATCC), an international depository of microorganismin USA, was examined in accordance with the DNA-DNA hybridization methodin Bergey's Manual of Systematic Bacteriology, Vol. 1 (1984). Twelvetype-strains shown in Table 1 in the below were respectively cultured ina usual manner, and proliferated cells were collected from the resultingcultures. Arthrobacter sp. S34, FERM BP-6450, was cultured by the seedculture method in the later described Example 2-1, followed bycollecting the proliferated cells. According to conventional method,DNAs were obtained from each type-strain of microorganisms, twomicrograms aliquots of the DNAs were digested with a restriction enzyme,Pst I. The resulting digested mixtures were respectively spotted on“Hybond-N+”, a nylon membrane commercialized by Amersham International,Arlington Heights, Ill., USA, and in a usual manner, treated withalkali, neutralized, and dried to fix the DNAs on the nylon membrane.One microgram of the DNA obtained from Arthrobacter sp. S34, FERMBP-6450, was provided and digested with Pst I. Using [α-³²P] dCTPcommercialized by Amersham International, Arlington Heights, Ill., USA,and “READY-TO-GO DNA-LABELLING KIT”, a DNA-labelling kit commercializedby Pharmacia LKB Biotechnology AB, Uppsala, Sweden, the digestant waslabelled with an isotope to obtain a probe. The probe and the above DNAfixed on nylon film were hybridized for two hours under shakingconditions at 65° C. in “RAPID HYBRIDIZATION BUFFER”, a buffer forhybridization commercialized by Amersham Corp., Div., AmershamInternational, Arlington Heights, Ill., USA. The nylon film afterhybridization washed in a usual manner, dried and subjected toautoradiography in a usual manner. Signals of hybridization observed onradiography were analyzed on “IMAGE MASTER”, an image analyzing systemcommercialized by Pharmacia LKB Biotechnology AB, Uppsala, Sweden,followed by expressing numerically the intensity of the signals forhybridization. Based on the numerals, the relative intensities (%) ofspots for the DNAs derived from the type-strains were calculated byregarding the signal intensity of a spot for the DNA from Arthrobactersp. S34, FERM BP-6450, as 100 and used as an index for the DNA homologybetween the microorganism and the type-strains. The results are in Table1.

TABLE 1 Signal intensity of Strain of microorganism hybridizationArthrobacter atrocyaneus, ATCC 13752 42.0 Arthrobacter aurescens, ATCC13344 12.4 Arthrobacter citreus, ATCC 11624 36.2 Arthrobactercrystallpoietes, ATCC 15481 31.6 Arthrobacter globiformis, ATCC 801055.1 Arthrobacter nicotianae, ATCC 15236 18.8 Arthrobacter oxydans, ATCC14358 28.3 Arthrobacter pascens, ATCC 13346 24.6 Arthrobacterprotophormiae, ATCC 19271 29.3 Arthrobacter ramosus, ATCC 13727 98.6Arthrobacter ureafaciens, ATCC 7562 42.3 Arthrobacter viscous, ATCC19584 0.0 Arthrobacter sp. S34, FERM BP-6450 100

As shown in Table 1, the signal intensity of hybridization for the spotof DNA from Arthrobacter ramosus type strain, ATCC 13727, was as high as98.6%. The data revealed that Arthrobacter sp. S34, FERM BP-6450, hadthe highest homology with Arthrobacter ramosus type-strain, ATCC 13727,among the 12 type strains used in this Example. The results in the aboveshows that Arthrobacter sp. S34, FERM BP-6450, is a novel microorganismnearly related to Arthrobacter ramosus type-strain, ATCC 13727.

EXAMPLE 2

Non-Reducing Saccharide-Forming Enzyme

Experiment 2-1

Preparation of Enzyme

A nutrient culture medium, consisting of 1.0 w/v % “PINE-DEX #4”, adextrin commercialized by Matsutani Chemical Ind., Tokyo, Japan, 0.5 w/v% peptone, 0.1 w/v % yeast extract, 0.1 w/v % monosodium phosphate, 0.06w/v % dipotassium hydrogen phosphate, 0.05 w/v % magnesium sulfate, andwater, was prepared and adjusted to pH 7.0. About 100 ml aliquots of themedium were placed in 500-ml Erlenmeyer flasks which were thenautoclaved at 120° C. for 20 min and cooled, followed by an inoculationof a seed of Arthrobacter sp. S34, FERM BP-6450 and a culture at 37° C.for 48 hours under stirring conditions of 260 rpm for obtaining a seedculture.

Except for containing 0.05 w/v % of “KM-75”, a antifoamer commercializedby Shin-Etsu Chemical, Co., Ltd, Tokyo, Japan, an about 20 l of the samenutrient culture medium as used in the seed culture was placed in a 30-lfermenter, sterilized, cooled to 37° C., and inoculated with one v/v %of the seed culture to the medium, followed by an incubation at 37° C.and pHs of 5.5-7.5 for about 72 hours under aeration-agitationconditions.

A portion of the resultant culture was sampled, centrifuged to separateinto cells and a culture supernatant. The cells were ultrasonicallydisrupted and centrifuged to collect supernatant for a cell extract.Assay for non-reducing saccharide-forming enzyme activity in eachculture supernatant and cell extract revealed that the former showed arelatively-low enzyme activity and the latter exhibited an about 0.1unit with respect to one milliliter of the culture.

EXAMPLE 2-2

Purification of Enzyme

An about 80 l of a culture, obtained according to the method in Example2-1, was centrifuged at 8,000 rpm for 30 min to obtain an about 800 gcells by wet weight. The wet cells were suspended in two liters of 10 Mphosphate buffer (pH 7.0) and treated with “MODEL UH-600”, an ultrasonichomogenizer commercialized by SMT Co., Tokyo, Japan. The resultingsolution was centrifuged at 10,000 rpm for 30 min to yield an about 2 lof a culture supernatant. To and in the culture supernatant was addedand dissolved ammonium sulfate to give a saturation degree of 0.7, andthe mixture was allowed to stand at 4° C. for 24 hours and centrifugedat 10,000 rpm for 30 min to obtain a precipitate. The precipitate thusobtained was dissolved in 10 mM phosphate buffer (pH 7.0) and dialyzedagainst a fresh preparation of the same buffer as above for 48 hours,followed by centrifuging the dialyzed inner solution at 10,000 rpm for30 min to remove insoluble substances. An about one liter of theresulting solution was subjected to an ion-exchange columnchromatography using a column packed with about 1.3 l of “SEPABEADSFP-DA13 GEL”, an anion exchanger commercialized by Mitsubishi ChemicalIndustries Ltd., Tokyo, Japan. The elution step was carried out using alinear gradient buffer of 10 mM phosphate buffer (pH 7.0) containingsalt which increased from 0 M to 0.6 M. The eluate from the column wasfractionated, and the fractions were respectively assayed fornon-reducing saccharide-forming enzyme activity. As a result, the enzymeactivity was remarkably found in fractions eluted with buffer having asalt concentration of about 0.2 M, followed by pooling the fractions.

Ammonium sulfate was added to the resulting solution to give aconcentration of 1 M, and the mixture was allowed to stand at 4° C. for12 hours, centrifuged at 10,000 rpm for 30 min to collect a supernatant.The supernatant thus obtained was subjected to hydrophobic columnchromatography using a column packed with “BUTYL TOYOPEARL 650M GEL”, ahydrophobic gel commercialized by Tosoh Corporation, Tokyo, Japan. Thegel volume used was about 300 ml and used after equilibrated with 10 mMphosphate buffer (pH 7.0) containing 1 M ammonium sulfate. The elutionstep was carried out using a linear gradient buffer of 10 mM phosphatebuffer (pH 7.0) containing ammonium sulfate which decreased from 1 M to0 M during the feeding. The eluate from the column was fractionated, andthe fractions were respectively assayed for non-reducingsaccharide-forming enzyme activity. As a result, the enzyme activity wasremarkably found in fractions eluted with buffer having a saltconcentration of about 0.75 M, followed by pooling the fractions.

The resulting solution was dialyzed against 10 mM phosphate buffer (pH7.0), and the resulting dialyzed inner solution was centrifuged at10,000 rpm for 30 min to collect a supernatant, followed by subjectingthe supernatant to ion-exchange column chromatography using a columnpacked with about 40 ml of “DEAE TOYOPEARL 650S GEL”, an anion exchangercommercialized by Tosoh Corporation, Tokyo, Japan. The elution step wascarried out using a linear aqueous salt solution which increased from 0M to 0.2 M during the feeding. The eluate from the column wasfractionated, and the fractions were respectively assayed fornon-reducing saccharide-forming enzyme activity. As a result, the enzymeactivity was remarkably found in fractions eluted with buffer having asalt concentration of about 0.15 M, followed by pooling the fractions.The resulting solution was further subjected to gel filtration columnchromatography using a column packed with about 380 ml of “ULTROGEL®AcA44 GEL”, a gel for gel filtration column chromatographycommercialized by Sepracor/IBF s.a. Villeneuve la Garenne, France,followed by collecting fractions with the desired enzyme activity. Thelevel of the non-reducing saccharide-forming enzyme activity, specificactivity, and yields in the above purification steps are in Table 2.

TABLE 2 Enzyme activity Specific of non-reducing activitysaccharide-forming (unit/mg Yield Purification step enzyme protein) (%)Cell extract 8,000 — 100 Dialyzed inner-solution 7,500 0.2 94 aftersalting out with ammonium salt Eluate from SEPABEADS 5,200 0.7 65 columnEluate from hydrophobic column 2,600 6.3 33 Eluate from TOYO PEARL 91067.4 11 Eluate of gel filtration 59.0 168 0.7

The solution eluted and collected from the above gel filtrationchromatography was in a usual manner subjected to electrophoresis using7.5 w/v % polyacrylamide gel and resulted in a single protein band. Thedata shows that the eluate from gel filtration chromatography was apurified specimen of a non-reducing saccharide-forming enzyme purifiedup to an electrophoretically homogeneous form.

EXAMPLE 2-3

Property of Enzyme

EXAMPLE 2-3(a)

Action

A 20% aqueous solution containing glucose, maltose, maltotriose,maltotetraose, maltopentaose, maltohexaose or maltoheptaose as asubstrate for enzyme was prepared, mixed with two units/g substrate,d.s.b., of a purified specimen of a non-reducing saccharide-formingenzyme obtained by the method in Example 2-2, and enzymatically reactedat 50° C. and pH 6.0 for 48 hours. The reaction mixture was desalted andanalyzed on high-performance liquid chromatography (abbreviated as“HPLC” hereinafter) using two columns of “MCI GEL CK04SS COLUMN”,commercialized by Mitsubishi Chemical Industries Ltd., Tokyo, Japan,which were cascaded in series, followed by determining the saccharidecomposition of the reaction mixture. The conditions and apparatus usedin HPLC were as follows: The column was kept at 85° C. using “Co-8020”,a column oven commercialized by Tosoh Corporation, Tokyo, Japan. Wateras a moving phase was fed at a flow rate of 0.4 ml/min. The eluate wasanalyzed on “RI-8020”, a differential refractometer commercialized byTosoh Corporation, Tokyo, Japan. The results were in Table 3.

TABLE 3 Elution time Percentage Substrate Reaction product (min) (%)Glucose Glucose 57.2 100.0 Maltose Maltose 50.8 100.0 MaltotrioseGlucosyltrehalose 43.2 36.2 Maltotriose 46.2 63.8 MaltotetraoseMaltosyltrehalose 38.9 87.2 Maltotetraose 42.3 12.8 MaltopentaoseMaltotriosyltrehalose 35.4 93.0 Maltopentaose 38.4 7.0 MaltohexaoseMaltotetraosyltrehalose 32.7 93.8 Maltohexaose 35.2 6.2 MaltoheptaoseMaltopentaosyltrehalose 30.2 94.2 Maltoheptaose 32.4 5.8

As evident form the results in Table 3, each reaction product consistedessentially of the remaining substrate and a newly formed non-reducingsaccharide of α-glucosyltrehalose, α-maltosyltrehalose,α-maltotriosyltrehalose, α-maltotetraosyltrehalose, orα-maltopentaosyltrehalose (in Table 3, it is expressed asglucosyltrehalose, maltosyltrehalose, maltotriosyltrehalose,maltotetraosyltrehalose, or maltopentaosyltrehalose). Substantially noother saccharide was detected in the reaction mixture. Regarding andevaluating the percentage of non-reducing saccharide in each reactionproduct as a production yield, it was revealed that the yield ofα-glucosyltrehalose having a glucose polymerization degree of 3 wasrelatively low and the yield of those having a glucose polymerizationdegree of 4 or higher such as α-maltosyltrehalose,α-maltotriosyltrehalose, α-maltotetraosyltrehalose, andα-maltopentaosyltrehalose was as high as about 85% or higher. Noformation of non-reducing saccharide from glucose and maltose wasobserved.

EXAMPLE 2-3(b)

Molecular Weight

A purified specimen of a non-reducing saccharide-forming enzyme,obtained by the method in Example 2-2, was subjected to SDS-PAGE using10 w/v % polyacrylamide gel in a usual manner in parallel with molecularmarkers commercialized by Japan Bio-Rad Laboratories, Tokyo, Japan.Comparing with the positions of the molecular markers afterelectrophoresis, the non-reducing saccharide-forming enzyme exhibited amolecular weight of about 75,000±10,000 daltons.

EXAMPLE 2-3(c)

Isoelectric Point

A purified specimen of a non-reducing saccharide-forming enzyme,obtained by the method in Example 2-2, was isoelectrophoresed using apolyacrylamide gel containing 2 w/v % “AMPHOLINE”, an ampholyte,commercialized by Pharmacia LKB Biotechnology AB, Uppsala, Sweden. Afterisoelectrophoresis, the measurement of the pH of gel revealed that thenon-reducing saccharide-forming enzyme had an isoelectric point of about4.5±0.5.

EXAMPLE 2-3(d)

Optimum Temperature and pH

Using a purified specimen of a non-reducing saccharide-forming enzyme,obtained by the method in Example 2-2, it was examined the influence oftemperature and pH on the activity of the non-reducingsaccharide-forming enzyme. When examining the influence of temperature,it was conducted similarly as in the assay for enzyme activity exceptfor reacting the enzyme at different temperatures. In the examination ofthe influence of pH, it was conducted similarly as in the assay forenzyme activity except for reacting the enzyme at different pHs usingappropriate 20 mM buffers. In each examination, a relative value (%) ofa lowered level of reducing power of substrate in each reaction systemwas calculated into its corresponding relative enzyme activity (%). FIG.1 shows a result of the influence of temperature, and FIG. 2 is of pH.The cross axles in FIGS. 1 and 2 show reaction temperatures and reactionpHs, respectively. As shown in FIG. 1, the optimum temperature of theenzyme was about 50° C. when incubated at pH 6.0 for 60 min. Also asshown in FIG. 2, the optimum pH of the enzyme was a pH of about 6.0 whenincubated at 50° C. for 60 min.

EXAMPLE 2-3(E)

Thermal and pH Stabilities

Using a purified specimen of a non-reducing saccharide-forming enzyme,obtained by the method in Example 2-2, it was examined the thermal andpH stabilities of the enzyme. The thermal stability was examined bydiluting the specimen with 20 mM phosphate buffer (pH 7.0), incubatingthe dilutions at prescribed temperatures for 60 min, cooling theincubated dilutions, and determining the remaining enzyme activity inthe dilutions according to the method of the assay for the enzymeactivity. The pH stability of the enzyme was examined by diluting thespecimen with 50 mM buffers with appropriate different pHs, incubatingthe dilutions at 4° C. for 24 hours, adjusting the dilutions to pH 6,and determining the remaining enzyme activity in the dilutions accordingto the method of the assay for the enzyme activity. The results of thethermal and pH stabilities of the enzyme are respectively shown in FIGS.3 and 4. The cross axles in FIGS. 3 and 4 show incubation temperaturesand pHs for the enzyme, respectively. As shown in FIG. 3, the enzyme wasstable up to about 55° C. and was stable at pHs in the range of about5.0 to about 10.0 as shown in FIG. 4.

These results evidence that the non-reducing saccharide forming-enzyme,obtained by the method in Example 2-2, is the present non-reducingsaccharide-forming enzyme having an optimum temperature in a mediumtemperature range.

EXAMPLE 2-4

Partial Amino Acid Sequence

A portion of a purified specimen of a non-reducing saccharide-formingenzyme, obtained by the method in Example 2-2, was dialyzed againstdistilled water to obtain an about 80 μg of a sample by weight as aprotein for analyzing the N-terminal amino acid sequence. Using “PROTEINSEQUENCER MODEL 473A”, a protein sequencer commercialized by AppliedBiosystems, Inc., Foster City, USA, the N-terminal amino acid sequencewas analyzed up to 20 amino acid residues from the N-terminus. Therevealed N-terminal amino acid sequence was the partial amino acidsequence of SEQ ID NO: 4. A portion of a purified specimen of anon-reducing saccharide-forming enzyme, obtained by the method inExample 2-2, was dialyzed against 10 mM Tris-HCl buffer (pH 9.0) and ina usual manner concentrated up to an about one mg/ml solution using“ULTRACENT-30”, an ultrafiltration membrane commercialized by TosohCorporation, Tokyo, Japan. To 0.2 ml of the concentrate was added 10 μg“TPCK-TRYPSIN”, a reagent trypsin commercialized by Wako Pure ChemicalIndustries, Ltd., Tokyo, Japan, allowed to react at 30° C. for 22 hoursto digest the enzyme to form peptides. The peptides were separated bysubjecting the reaction mixture to reverse-phase HPLC using “μBONDASPHERE C18 COLUMN” having a diameter of 3.9 mm and a length of 150mm, a product of Waters Chromatography Div., MILLIPORE Corp., Milford,USA. The elution step was carried out at ambient temperature by feedingto the column an aqueous solution containing 0.1 v/v % trifluoro acetateand acetonitrile increasing from 24 to 48 v/v % for 60 min during thefeeding at a flow rate of 0.9 ml/min. The peptides eluted from thecolumn were detected by monitoring the absorbance at a wavelength of 210nm. Two peptides, which were well separated from others, i.e., “S5”eluted at a retention time of about two hours and “S8” eluted at aretention time of about 30 min were separated, respectively dried invacuo, and dissolved in 50 v/v % aqueous acetonitrile solutionscontaining 50 μl of 0.1 v/v % trifluoro acetate. The peptide solutionswere subjected to the protein sequencer to analyze up to 20 amino acidresidues. From peptides “S5” and “S8” the amino acid sequences of SEQ IDNos:5 and 6 were obtained.

EXAMPLE 3

DNA Encoding Non-Reducing Saccharide-Forming Enzyme

EXAMPLE 3-1

Construction and Screening of Gene Library

Except for setting temperature and time for culture were respectivelyset to 27° C. and 24 hours, Arthrobacter sp. S34, FERM BP-6450, wascultured similarly as in Example 2-1.

The culture was centrifuged to remove cells which were then suspended inan adequate amount of Tris-EDTA-salt buffered saline (hereinafterdesignated as “TES buffer”) (pH 8.0), admixed with lysozyme in an amountof 0.05 w/v % to the cell suspension by volume, followed by anincubation at 37° C. for 30 min. The resultant mixture was freezed bystanding at −80° C. for one hour, and then admixed and sufficientlystirred with a mixture of TES buffer and phenol preheated to 60° C.,cooled, and centrifuged to collect the formed supernatant. To thesupernatant was added cold ethanol was added, and then the formedsediment was collected, dissolved in an adequate amount of SSC buffer(pH 7.1), admixed with 7.5 μg ribonuclease and 125 μg protease, andincubated at 37° C. for one hour. The resulting mixture was admixed andstirred with chloroform/isoamyl alcohol, and allowed to stand, followedby collecting the formed upper layer, adding cold ethanol to the layer,and collecting the formed sediment. The sediment was rinsed with a cold70 v/v ethanol, dried in vacuo to obtain a DNA, followed by dissolvingin SSC buffer (pH 7.1) to give a concentration of about one mg/ml, andfreezing at −80° C.

Fifty microliters of the DNA was provided, admixed with an abut 50 unitsof KpnI as an restriction enzyme, and incubated at 37° C. for one hourto digest the DNA. Three micrograms of the digested DNA and 0.3microgram of “pBluescript II SK (+)”, a plasmid vector commercialized byStratagene Cloning Systems, California, USA, was weighed, subjected tothe action, were ligated using “DNA LIGATION KIT”, commercialized byTakara Shuzo Co., Ltd., Tokyo, Japan, according to the protocol affixedto the kit. According to conventional competent cell method, 100 μl of“Epicurian Coli XL1-Blue”, an Escherichia coli strain commercialized byStratagene Cloning Systems, California, USA, was transformed with theligated product. Thus a gene library was obtained.

The gene library thus obtained was inoculated to a agar nutrient platemedium (pH 7.0) containing 10 g/l tryptone, 5 g/l yeast extract, 5 g/lsodium chloride, 75 mg ampicillin sodium salt, and 50 mg/l5-bromo-4-chloro-indolyl-β-galactoside, and incubated at 37° C. for 18hours. About 5,000 white colonies formed on the medium were in a usualmanner fixed on “HYBOND-N-+”, a nylon film commercialized AmershamCorp., Div. Amersham International, Arlington Heights, Ill., USA. Basedon 1-8 amino acid residues in the amino acid sequence of SEQ ID NO: 5revealed in Example 2-4, an oligonucleotide having the nucleotidesequence of SEQ ID NO: 18 was chemically synthesized, and in a usualmanner labelled with [γ-³²P] ATP and T4 polynucleotide kinase to obtaina probe. Using the probe, the colonies, which had been fixed on thenylon film and obtained previously, were screened by conventional colonyhybridization method. The hybridization was carried out at 65° C. for 16hours in a solution for hybridization containing 6×SSC, 5× Denhaltsolution, and 100 mg/l of denatured salmon sperm DNA. The above nylonfilm after the hybridization washed with 6×SSC at 65° C. for 30 min, andfurther washed with 2×SSC containing 0.1 w/v % SDS at 65° C. for twohours. The resulting nylon film was in a usual manner subjected toautoradiography, and then, based on the signals observed on theautoradiography, a colony which strongly hybridized with the probe wasselected and named “GY1” as a transformant.

EXAMPLE 3-2

Decoding of Nucleotide Sequence

According to conventional manner, the transformant GY1 was inoculated toL-broth (pH 7.0) containing 100 μg/ml ampicillin in a sodium form, andcultured at 37° C. for 24 hours under shaking conditions. Aftercompletion of the culture, the proliferated cells were collected fromthe culture by centrifugation and treated with conventional alkali-SDSmethod to extract a recombinant DNA. The recombinant DNA was named pGY1.Using the above probe, the recombinant DNA, pGY1, was analyzed onconventional Southern blot technique, and based on the analytical data arestriction map was constructed as shown in FIG. 5. As shown in FIG. 5,it was revealed that the recombinant DNA, pGY1, contained a nucleotidesequence consisting of bases of about 5,500 base-pairs (bp) fromArthrobacter sp. S34, FERM BP-6450, expressed with a bold line, and thatthe recombinant DNA contained a nucleotide sequence encoding the presentnon-reducing saccharide-forming enzyme, as indicated with a black arrowwithin the area of the bold line, in the area consisting bases of about4,000 bp between two recognition sites by a restriction enzyme, EcoRI.Based on the result, the recombinant DNA, pGY1, was completely digestedwith EcoRI, and then a DNA fragment of about 4,000 bp was separated andpurified using conventional agarose gel electrophoresis. The DNAfragment and “pBluescript II SK (+)”, a plasmid vector commercialized byStratagene Cloning Systems, California, USA, which had been previouslydigested with EcoRI, were ligated with conventional ligation method.With the ligated product, “XL1-BLUE”, an Escherichia coli straincommercialized by Stratagene Cloning Systems, California, USA, wastransformed to obtain a transformant. A recombinant DNA was extractedfrom the transformant in a usual manner, confirming in a usual mannerthat it contained the aforesaid DNA fragment consisting of about basesof 4,000 bp, and named it “pGY2”. The transformant introduced with“pGY2” was named “GY2”.

The analysis of the nucleotide sequence of the recombinant DNA pGY2 onconventional dideoxy method revealed that it contained the nucleotidesequence of SEQ ID NO: 19 consisting bases of 3252 bp derived fromArthrobacter sp. S34, FERM BP-6450. The nucleotide sequence encodes theamino acid sequence as shown in parallel in SEQ ID NO: 19. Comparing theamino acid sequences of SEQ D NOs: 4 to 6 as partial amino acidsequences of the present non-reducing saccharide-forming enzymeconfirmed in Example 2-4, the amino acid sequences of SEQ ID Nos:4 to 6were perfectly coincided with the amino acids 2-21, 619-638, and 98-117in SEQ ID NO: 19. These data indicate that the present non-reducingsaccharide-forming enzyme obtained in Example 2 consists of the aminoacids 2-757 of SEQ ID NO: 19, or has the amino acid Sequence of SEQ IDNO: 1, and that the enzyme of Arthrobacter sp. S34, FERM BP-6450, isencoded by a nucleotide sequence of bases 746-3013 of SEQ ID NO: 19, orencoded by the nucleotide sequence of SEQ ID NO: 7. The structure of therecombinant DNA pGY2 is in FIG. 6.

The above-identified amino acid sequence of the present non-reducingsaccharide-forming enzyme obtained by the method in Example 2, and aminoacid sequences of known enzymes having a non-reducing saccharide-formingactivity were compared using “GENETYX-MAC, VER. 8”, a commerciallyavailable computer program commercialized by Software Development Co.,Ltd., Tokyo, Japan, according to the method by Lipman, David J. inScience, Vol. 227, pp. 1,435-1,441 (1985) to calculate their homology(%). The enzymes used as known enzymes were those from Arthrobacter sp.Q36 and Rhizobium sp. M-11 disclosed in Japanese Patent Kokai No.322,883/95; Sulfolobus acidocaldarius, ATCC 33909, disclosed in JapanesePatent Kokai No. 84,586/96; and Sulfolobus solfataricus KM1 disclosed inSai-Kohyo No. WO 95/34642. As disclosed in the above publications, theconventional enzymes have optimum temperatures other than a mediumtemperature range. The information of amino acid sequences ofconventional enzymes is obtainable from the GeneBank, a DNA databaseproduced by the National Institutes of Health (NIH), USA, under theaccession numbers of D63343, D64128, D78001 and D83245. The obtainedhomologies are in Table 4.

TABLE 4 Origin of enzyme for amino acid Homology on amino acidsequence(*) comparison sequence Rhizobium sp. M-11 (D78001) 56.9%Arthrobacter sp. Q36 (D63343) 56.6% Sulfolobus solfataricus KM1 33.2%(D64128) Sulfolobus acidocaldarius, 31.4% ATCC 33909 (D83245) (*):Numerals in parentheses are access numbers to the GeneBank.

As shown in Table 4, the present non-reducing saccharide-forming enzymein Example 2 showed a highest amino acid homology of 56.9% with theenzyme from Rhizobium sp. M-11 among conventional enzymes with optimumtemperatures out of a medium temperature range. The data indicates thatthe present non-reducing saccharide-forming enzyme generally comprisesan amino acid sequence with a homology of at least 57% with the aminoacid sequence of SEQ ID NO: 1. The comparison result on amino acidsequence revealed that the enzyme in Example 2 and the above-identifiedfour types of conventional enzymes have common amino acid sequences ofSEQ ID NOs: 2 and 3. The enzyme in Example 2 has partial amino acidsequences of SEQ ID NOs: 2 and 3 as they correspond to amino acids 84-89and 277-282 in SEQ ID NO: 1. The four types of enzymes used asreferences have the above partial amino acid sequences which arepositioned at their corresponding parts. Based on the fact that any ofthe present enzyme in Example 2 and the enzymes as references have acommon activity of forming non-reducing saccharides having a trehalosestructure as an end unit from reducing partial starch hydrolysates, itwas indicated that the partial amino acid sequences of SEQ ID NOs: 2 and3 correlated to the expression of such an enzyme activity. These resultsshow that the present non-reducing saccharide-forming enzyme can becharacterized in that it comprises the amino acid sequences of SEQ IDNOs: 2 and 3, and has an optimum temperature in a medium temperaturerange.

EXAMPLE 3-3

Transformant Introduced with DNA

Based on the 5′- and 3′-termini of the nucleotide sequence of SEQ ID NO:7, an oligonucleotide of the nucleotide sequences of SEQ ID NOs: 20 and21 were chemically synthesized in a usual manner. As sense- andanti-sense-primers, 85 ng of each of the oligonucleotide and 100 ng ofthe recombinant DNA pGY2 in Example 3-2 as a template were mixed in areaction tube, and the mixture was admixed with 1.25 units of“PYROBEST”, a thermostable DNA polymerase specimen commercialized byTakara Shuzo Co., Ltd., Tokyo, Japan, together with 5 μl of a bufferaffixed with the specimen and 4 μl of a dNTP mixture. The resultingmixture was brought up to a volume of 50 μl with sterilized distilledwater to effect PCR. The temperature for PCR was controlled in such amanner that the mixture was treated with 25 cycles of successiveincubations of 95° C. for one minute, 98° C. for 20 seconds, 70° C. for30 seconds, and 72° C. for four minutes, and finally incubated at 72° C.for 10 min. A DNA as a PCR product was collected in a usual manner toobtain an about 2,300 bp DNA. The DNA thus obtained was admixed with“pKK223-3”, a plasmid vector commercialized by Pharmacia LKBBiotechnology AB, Uppsala, Sweden, which had been previously cleavedwith a restriction enzyme, EcoRI, and blunted by “DNA BLUNTING KIT”commercialized by Takara Shuzo Co., Ltd., Tokyo, Japan, and ligated byconventional ligation method. Thereafter, the ligated product wastreated in a usual manner to obtain a recombinant DNA introduced withthe above DNA consisting of bases of about 2,300 bp. Decoding of therecombinant DNA showed that it comprised a nucleotide sequence which twonucleotide sequences of 5′-ATG-3′ and 5′-TGA-3′ were respectively addedto the 5′- and 3′-termini of the nucleotide sequence of SEQ ID NO: 7.The DNA was named “pGY3”. The structure of the recombinant DNA pGY3 wasin FIG. 7.

The recombinant DNA pGY3 was in a usual manner introduced into anEscherichia Coli LE 392 strain, ATCC 33572, which had been competentedin conventional manner, to obtain a transformant. Conventionalalkali-SDS method was applied for the transformant to extract a DNA, andthen the extracted DNA was confirmed to be pGY3 in a usual manner andnamed “GY3”. Thus a transformant introduced with a DNA encoding thepresent non-reducing saccharide-forming enzyme.

EXAMPLE 3-4

Transformant Introduced with DNA

Based on a nucleotide sequence in the downstream of the 3′-terminus of apromotor in “pKK223-3”, a plasmid vector commercialized by Pharmacia LKBBiotechnology AB, Uppsala, Sweden, oligonucleotide having the nucleotidesequences of SEQ ID NOs: 22 and 23 were synthesized in conventionalmanner, and phosphorylated their 5′-termini using T4 polynucleotidekinase. The phosphorylated oligonucleotide were annealed, ligated with“pKK223-3”, a plasmid vector commercialized by Pharmacia LKBBiotechnology AB, Uppsala, Sweden, which had been previously cleavedwith restriction enzymes of EcoRI and PstI, by conventional ligationmethod. According to conventional method, the ligated product wasintroduced into an Escherichia coli strain which was then cultured andtreated with alkali-SDS method to extract a DNA. The DNA thus obtainedhad a similar structure to a plasmid vector “pKK223-3”, and hadrecognition sites by restriction enzymes of EcoRI, XbaI, SpeI, and PstIat the downstream of the promoter. The present inventors named the DNA aplasmid vector “pKK4”.

Similarly as in Example 3-3, PCR was done except for usingoligonucleotide with the nucleotide sequences of SEQ ID NOs: 24 and 25,which had been chemically synthesized based on the 5′- and 3′-terminalpartial nucleotide sequences of SEQ ID NO: 7. A DNA as a PCR product wascollected in a usual manner to obtain an about 2,300 bp DNA. The DNAthus obtained was cleaved with restriction enzymes, XbaI and SpeI, andthe above plasmid vector pKK4, which had been cleaved with XbaI andSpeI, were ligated by conventional ligation method. Thereafter, theligated product was treated in a usual manner to obtain a recombinantDNA with the nucleotide sequence of SEQ ID NO: 7. The recombinant DNAwas named “pKGY1”.

Using overlap extension method, which two steps PCR were applied for andreported by Horthon, Robert M. in Methods in Enzymology, Vol. 217, pp.270-279 (1993), a nucleotide sequence in the upper part of the5′-terminus of SEQ ID NO: 7 in the above DNA pKGY1 was modified. PCR asa first step PCR-A was done similarly as in Example 3-3 except forusing, as sense- and anti-sense-primers, oligonucleotide of thenucleotide sequences of SEQ ID NOs: 26 and 27, which had been chemicallysynthesized based on the nucleotide sequence of plasmid vector pKK4; andas a template the above recombinant DNA pKGY1. In parallel, PCR as afirst step PCR-B was done similarly as in Example 3-3 except for using,as sense- and anti-sense-primers, oligonucleotide of the nucleotidesequences of SEQ ID NOs: 28 and 29, which had been respectivelychemically synthesized in a usual manner based on the nucleotidesequence of SEQ ID NO: 7; and as a template the above recombinant DNApKGY1. A DNA as a product of the first step PCR-A was collected in ausual manner to obtain an about 390 bp DNA. A DNA as a product in thefirst stp PCR-B was collected in conventional manner to obtain an about930 bp DNA.

PCR, as a second step PCR-A, was done similarly as in Example 3-3 exceptfor using as a template a DNA mixture, i.e., a product of the firstPCR-A and the first step PCR-B; as a sense primer the oligonucleotidesequence of the nucleotide sequence of SEQ ID NO: 26; and as ananti-sense primer the oligonucleotide of the nucleotide sequence of SEQID NO: 30, which had been chemically synthesized in conventional mannerbased on the nucleotide sequence of SEQ ID NO: 7. The DNA as a productin the PCR was collected in a usual manner to obtain an about 1,300 bpDNA.

The DNA as a product in the second PCR-A was cleaved with restrictionenzymes of EcoRI and BsiWI, and the formed DNA consisting of bases ofabout 650 bp was collected in a usual manner. An about 6,300 bp DNA,which was formed after cleavage of the above recombinant DNA pKGYl withrestriction enzymes of EcoRI and BsiWI, was collected in conventionalmanner. These DNAs were ligated in a usual manner, and the ligatedproduct was treated in conventional manner to obtain a recombinant DNAcomprising an about 650 bp DNA derived from the second step PCR-A.Decoding of the DNA by conventional dideoxy method revealed that theobtained recombinant DNA comprised a nucleotide sequence which thenucleotide sequence of SEQ ID NO: 8, a nucleotide sequence representedby 5′-ATG-3′, and a nucleotide sequence represented by 5′-TGA-3′ werecascaded in the order as indicated above from the 5′-terminus to the3′-terminus. The recombinant DNA thus obtained was named “pGY4”. Thestructure of pGY4 is substantially the same as the recombinant DNA pGY3except for that pGY4 comprises the nucleotide sequence of SEQ ID NO: 8.

The recombinant DNA pGY4 was introduced in conventional manner with“BMH71-18mutS”, an Escherichia coli competent cell commercialized byTakara Shuzo Co., Ltd., Tokyo, Japan to obtain a transformant. Thetransformant was treated with alkali-SDS method to extract a DNA whichwas then identified with pGY4 in conventional manner. Thus atransformant introduced with a DNA encoding the present non-reducingsaccharide-forming enzyme.

EXAMPLE 4

Preparation of Non-Reducing Saccharide-Forming Enzyme

EXAMPLE 4-1

Preparation of Enzyme Using Microorganism Of the Genus Arthrobacter

In accordance with the method in Example 2-1, Arthrobacter sp. S34, FERMBP-6450, was cultured by a fermenter for about 72 hours. After thecultivation, the resulting culture was concentrated with an SF-membraneto yield an about eight liters of a cell suspension. The cell suspensionwas treated with “MINI-LABO”, a supper high-pressure cell disruptercommercialized by Dainippon Pharmaceutical Co., Ltd., Tokyo, to disruptthe cells. The resulting solution was centrifuged to obtain an about 8.5l of a supernatant. When measured for non-reducing saccharide-formingactivity in the supernatant, it showed an about 0.1 unit of the enzymeactivity with respect to one milliliter of the culture. Ammonium sulfatewas added to the supernatant to brought up to a saturation degree ofabout 0.7 to salt out, and the sediment was collected by centrifugation,dissolved in 10 mM phosphate buffer (pH 7.0), and dialyzed against afresh preparation of the same buffer. Except for using an about 2 l ofan ion-exchange resin, the resulting dialyzed inner solution was fed toion-exchange column chromatography using “SEPABEADS FP-DA13 GEL”, ananion exchanger commercialized by Mitsubishi Chemical Industries Ltd.,Tokyo, Japan, as described in Example 2-2, to collect fractions withnon-reducing saccharide-forming enzyme. The fractions were pooled,dialyzed against a fresh preparation of the same buffer but containing 1M ammonium sulfate, and the resulting dialyzed inner solution wascentrifuged to collect the formed supernatant. Except for using an about300 ml gel, the supernatant was fed to hydrophobic column chromatographyin accordance with the method described in Example 2-2 to collectfractions with non-reducing saccharide-forming enzyme. Then it wasconfirmed that the obtained enzyme had an optimum temperature over 40°C. but below 60° C., i.e., a temperature in a medium temperature range,and an acid pH range of less than 7.

Thus an about 2,600 units of the present non-reducing saccharide-formingenzyme was obtained.

EXAMPLE 4-2

Preparation of Enzyme Using Transformant

One hundred ml of an aqueous solution containing 16 g/l polypeptone, 10g/l yeast extract, and 5 g/l sodium chloride was placed in a 500-mlErlenmeyer flask, autoclaved at 121° C. for 15 min, cooled, adjustedaseptically to pH 7.0, and admixed aseptically with 10 mg of ampicillinin a sodium salt to obtain a liquid nutrient medium. The nutrient mediumwas inoculated with the transformant GY2 in Example 3-2, and incubatedat 37° C. for about 20 hours under aeration-agitation conditions toobtain a seed culture. Seven liters of a medium having the samecomposition as used in the seed culture was prepared as in the case ofthe seed culture and placed in a 10-l fermenter, and inoculated with 70ml of the seed culture, followed by the incubation for about 20 hoursunder aeration-agitation conditions. From the resultant culture cellswere collected by centrifugation in a usual manner. The collected cellswere suspended in phosphate buffer (pH 7.0), disrupted by the treatmentof ultrasonication, and centrifuged to remove insoluble substances,followed by collecting a supernatant to obtain a cell extract. Theextract was dialyzed against 10 mM phosphate buffer (pH 7.0). Theresulting dialyzed inner solution was collected and confirmed that itexhibited a non-reducing saccharide-forming enzyme activity, had anoptimum temperature in a medium temperature range, i.e., a temperatureof over 40° C. but below 60° C., and had an optimum pH in an acid pHrange, i.e., a pH of less than 7.

Thus the present non-reducing saccharide-forming enzyme was obtained. Inthe culture of this example, an about 0.2 unit/ml culture of the enzymewas produced.

As a control, “XL1-BLUE”, an Escherichia coli strain commercialized byStratagene Cloning Systems, California, USA, was cultured under the sameconditions as above in a nutrient culture medium of the same compositionas used in the above except that it contained no ampicillin. Similarlyas above, a cell extract was obtained and dialyzed. No activity ofnon-reducing saccharide-forming enzyme was detected in the resultingdialyzed inner solution, meaning that the transformant GY2 is useful inproducing the present non-reducing saccharide-forming enzyme.

EXAMPLE 4-3

Preparation of Enzyme Using Transformant

The transformant GY3 in Example 3-3 was cultured similarly as in Example4-2 except for using a liquid nutrient culture medium consisting of onew/v % maltose, three w/v polypeptone, one w/v % “MEAST PIG”, a productof Asahi Breweries, Ltd., Tokyo, Japan, 0.1 w/v % dipotassium hydrogenphosphate, 100 μg/ml ampicillin, and water. The resultant culture wastreated with ultrasonication to disrupt cells, and the resulting mixturewas centrifuged to remove insoluble substances. When assayed fornon-reducing saccharide-forming enzyme activity in the resultingsupernatant, the culture contained about 15 units/ml culture of theenzyme. In accordance with the method in Example 2-2, the enzyme in thesupernatant was purified, confirming that the resulting purifiedspecimen exhibited a non-reducing saccharide-forming enzyme activity,had an optimum temperature in a medium temperature range, i.e., atemperature of over 40° C. but below 60° C., and had an optimum pH in anacid pH range, i.e., a pH of less than 7. Thus the present non-reducingsaccharide-forming enzyme was obtained.

EXAMPLE 4-4

Preparation of Enzyme Using Transformant

The transformant GY4 in Example 3-4 was cultured similarly as in Example4-2 except for using a liquid nutrient culture medium consisting of twow/v % maltose, four w/v % peptone, one w/v % yeast extract, 0.1 w/v %sodium dihydrogen phosphate, 200 μg/ml ampicillin, and water. Theresultant culture was treated with ultrasonication to disrupt cells, andthe resulting mixture was centrifuged to remove insoluble substances.When assayed for non-reducing saccharide-forming enzyme activity in theresulting supernatant, the culture contained about 60 units/ml cultureof the enzyme. In accordance with the method in Example 2-2, the enzymein the supernatant was purified, confirming that the resulting purifiedspecimen exhibited a non-reducing saccharide-forming enzyme activity,had an optimum temperature in a medium temperature range, i.e., atemperature of over 40° C. but below 60° C., and had an optimum pH in anacid pH range, i.e., a pH of less than 7. Thus the present non-reducingsaccharide-forming enzyme was obtained.

EXAMPLE 5

Trehalose-Releasing Enzyme

EXAMPLE 5-1

Production of Enzyme

According to the method in EXample 2-1, Arthrobacter sp. S34, FERMBP-6450, was cultured by a fermenter. Then, in accordance with themethod in Example 2-2, the resulting culture was sampled, followed byseparating the sample into cells and a supernatant. From the cells acell extract was obtained. When assayed for trehalose-releasing activityof the supernatant and the cell extract, the former scarcely exhibitedthe enzyme activity, while the latter exhibited an about 0.3 uni/mlculture of the enzyme.

EXAMPLE 5-2

Preparation of Enzyme

An about 80 l of a culture, prepared according to the method in Example2-1, was centrifuged at 8,000 rpm for 30 min to obtain an about 800 gcells by wet weight. Two l of the wet cells was suspended in 10 mMphosphate buffer (pH 7.0) and treated with “MODEL UH-600”, an ultrasonichomogenizer commercialized by MST Co., Tokyo, Japan. The resultingsuspension was centrifuged at 10,000 rpm for 30 min, followed acollection of an about two liters of a supernatant. The supernatant wasadmixed with ammonium sulfate to bring to a saturation degree of 0.7,allowed to stand at 4° C. for 24 hours, and centrifuged at 10,000 rpmfor 30 min to obtain a precipitate salted out with ammonium sulfate. Theprecipitate was dissolved in 10 mM phosphate buffer (pH 7.0), dialyzedagainst a fresh preparation of the same buffer for 48 hours, andcentrifuged at 10,000 rpm for 30 min to remove insoluble substances. Anabout one liter of the resulting dialyzed inner solution was fed toion-exchange column chromatography using an about 1.3 l of “SEPABEADSFP-DA13 GEL”, an anion exchanger commercialized by Mitsubishi ChemicalIndustries Ltd., Tokyo, Japan. The elution step was carried out using alinear 10 mM phosphate buffer (pH 7.0) containing salt decreasing from 0M to 0.6 M during the feeding. The eluate from the column wasfractionated, and the fractions each were assayed fortrehalose-releasing enzyme activity. As a result, the enzyme activitywas remarkably found in fractions eluted with buffer having a saltconcentration of about 0.2 M, followed by pooling the fractions.

Ammonium sulfate was added to the pooled solution to bring to aconcentration of 1 M, and the mixture was allowed to stand at 4° C. for12 hours, centrifuged at 10,000 rpm for 30 min to collect a supernatant.The supernatant was subjected to hydrophobic column chromatography usinga column packed with “BUTYL TOYOPEARL 650M GEL”, a hydrophobic gelcommercialized by Tosoh Corporation, Tokyo, Japan. Prior to use, the gelvolume was set to about 300 ml and equilibrated with 10 mM phosphatebuffer (pH 7.0) containing 1 M ammonium sulfate. The elution step wascarried out using a linear gradient aqueous solution of ammoniumdecreasing from 1 M to 0 M during the feeding. The eluate from thecolumn was fractionated, and the fractions were respectively assayed fortrehalose-releasing enzyme activity. As a result, the enzyme activitywas remarkably found in fractions eluted with buffer having an ammoniumconcentration of about 0.5 M, followed by pooling the fractions.

The fractions were pooled, dialyzed against 10 mM phosphate buffer (pH7.0), and the dialyzed inner solution was centrifuged at 10,000 rpm for30 min. Then the resulting supernatant was collected and subjected to“DEAE-TOYOPEARL 650S GEL”, an anion exchanger commercialized by TosohCorporation, Tokyo, Japan. The elution step was carried out using alinear gradient aqueous solution of salt increasing from 0 M to 0.2 Mduring the feeding. The eluate from the column was fractionated, and thefractions were respectively assayed for trehalose-releasing enzymeactivity. As a result, the enzyme activity was remarkably found infractions eluted with buffer having an ammonium concentration of about0.15 M, followed by pooling the fractions. The pooled solution wassubjected to gel filtration chromatography using about 380 ml of“ULTROGEL® AcA44 RESIN”, a gel for gel filtration column chromatographycommercialized by Sepracor/IBF s.a. Villeneuve la Garenne, France,followed collecting fractions with a remarkable activity of the enzyme.The content, specific activity, and yield of the enzyme in each step arein Table 5.

TABLE 5 Activity of Trehalose- Specific releasing activity Yield Stepenzyme (unit) (mg/protein) (%) Cell extract 24,000 — 100 Dialyzed innersolution 22,500 0.6 94 after salting out with ammonium sulfate Eluatefrom SEPABEADS column 15,600 2.0 65 Eluate from hydrophobic column 6,40025.3 27 Eluate from TOYOPEARL column 4,000 131 17 Eluate after gelfiltration 246 713 1.0

When electrophoresed in 7.5 w/v % polyacrylamide gel in conventionalmanner, the solution eluted and collected from the above gel filtrationchromatography gave a single protein band. The data indicates that theeluate from gel filtration chromatography obtained in the above was apurified trehalose-releasing enzyme purified up to anelectrophoretically homogeneous level.

EXAMPLE 5-3

Property of Enzyme

EXAMPLE 5-3(a)

Action

Any one of saccharides consisting of α-glucosyltrehalose,α-maltosyltrehalose, α-maltotriosyltrehalose, α-maltotetraosyltrehalose,and α-maltopentaosyltrehalose as non-reducing saccharides having atrehalose structure obtained by the method in the later describedExample 8-3; and maltotriose, maltotetraose, maltopentaose,maltohexaose, and maltoheptaose as reducing saccharides was dissolved inwater into a 2 w/v % solution as an aqueous substrate solution forsubstrate. Each aqueous substrate solution was admixed with two units/gsubstrate, d.s.b., of a purified specimen of trehalose-releasing enzymeobtained by the method in Example 5-2, and enzymatically reacted at 50°C. and pH 6.0 for 48 hours. In accordance with the method in Example2-3(a), the reaction product was analyzed on HPLC after desalting tocalculate the saccharide composition of the reaction products each. Theresults are in Table 6. In Table 6, α-glucosyltrehalose,α-maltosyltrehalose, α-maltotriosyltrehalose, α-maltotetraosyltrehalose,and α-maltopentaosyltrehalose were respectively expressed asglucosyltrehalose, maltosyltrehalose, maltotriosyltrehalose,maltotetraosyltrehalose, and maltopentaosyltrehalose.

TABLE 6 Elution Compo- time sition Substrate Reaction product (min) (%)Glucosyltrehalose Trehalose 48.5 16.8 Glucose 57.2 8.2 Glucosyltrehalose43.3 75.0 Maltosyltrehalose Trehalose 48.5 44.1 Maltose 50.8 44.4Maltosyltrehalose 38.9 11.5 Maltotriosyltrehalose Trehalose 48.5 40.5Maltotriose 46.2 59.0 Maltotriosyltrehalose 35.4 0.5Maltotetraosyltrehalose Trehalose 48.5 35.0 Maltotetraose 42.1 64.2Maltotetraosyltrehalose 32.7 0.3 Maltopentaosyltrehalose Trehalose 48.529.5 Maltopentaose 38.2 70.2 Maltopentaosyltrehalose 30.2 0.3Maltotriose Maltotriose 46.2 100.0 Maltotetraose Maltotetraose 42.1100.0 Maltopentaose Maltopentaose 38.2 100.0 Maltohexaose Maltohexaose35.2 100.0 Maltoheptaose Maltoheptaose 32.6 100.0

As evident from the results in Table 6, the trehalose-releasing enzyme,obtained by the method in Example 5-2, specifically hydrolyzed anon-reducing saccharide, which has a trehalose structure as an end unitand a glucose polymerization degree of at least three, to releasetrehalose from the rest of the non-reducing saccharide to form trehaloseand a reducing saccharide having a glucose polymerization degree of oneor more. While the enzyme did not act on maltooligosaccharides such asmaltotriose and lower saccharides.

EXAMPLE 5-3(b)

Molecular Weight

A purified specimen of a trehalose-releasing enzyme, obtained by themethod in Example 5-2, was subjected along with molecular markerscommercialized by Japan Bio-Rad Laboratories, Tokyo, Japan, toconventional SDS-PAGE using 10 w/v % polyacrylamide gel. Afterelectrophoresis, the position of the specimen electrophoresed on the gelwas compared with those of the markers, revealing that the specimen hada molecular weight of about 62,000±5,000 daltons.

EXAMPLE 5-3(c)

Isoelectric Point

A purified specimen of a trehalose-releasing enzyme, obtained by themethod in Example 5-2, was in a usual manner subjected toisoelectrophoresis using a polyacrylamide gel containing 2 w/v %“AMPHOLINE”, an ampholyte commercialized by Pharmacia LKB BiotechnologyAB, Uppsala, Sweden. Measurement of pH of the gel after electrophoresis,it had an isoelectric point of about 4.7±0.5.

EXAMPLE 5-3(d)

Optimum Temperature and pH

A purified specimen of a trehalose-releasing enzyme, obtained by themethod in Example 5-2, was examined on the influence of the temperatureand pH on the enzyme activity. The influence of temperature was examinedaccording to the assay for enzyme activity except for reacting theenzyme at different temperatures. The influence of pH was examinedaccording to the assay for enzyme activity except for reacting theenzyme at different pHs using appropriate 20 mM buffers. In eachprocedure, relative values (%) of the increased level of reducing powerfound in each system were calculated and regarded as relative enzymeactivity (%). The results of the influence of temperature and pH arerespectively in FIGS. 8 and 9. The cross axles in FIGS. 8 and 9 showreaction temperatures and pHs for the enzyme, respectively. As shown inFIG. 8, the optimum temperature of the enzyme was about 50 to about 55°C. when incubated at pH 6.0 for 30 min, while the optimum pH of theenzyme was a pH of about 6.0 when incubated at 50° C. for 30 min.

EXAMPLE 5-3(e)

Stability on Temperature and pH

A purified specimen of a trehalose-releasing enzyme, obtained by themethod in Example 5-2, was examined on the stability of temperature andpH. The stability of temperature was examined by diluting the specimenwith 20 mM phosphate buffer (pH 7.0), incubating the dilutions atdifferent temperatures for 60 min, cooling the resulting dilutions, andassaying the enzyme activity remained in the dilutions. The pH stabilitywas studied by diluting the specimen with 50 mM buffers (pH 7.0) withdifferent pHs, incubating the dilutions at 4° C. for 24 hours, adjustedto pH 6, and assaying the enzyme activity remained in the dilutions. Theresults of the stability of temperature and pH are respectively in FIGS.10 and 11. The cross axles in FIGS. 10 and 11 show temperatures and pHsat which the enzyme was kept, respectively. As shown in FIG. 10, theenzyme was stable up to about 50° C., while the enzyme was stable at pHsin the range of about 4.5 to about 10.0.

The results described hereinbefore indicate that the trehalose-releasingenzyme, obtained by the method in Example 5-2, is the present enzymewhich has an optimum temperature in a medium temperature range.

EXAMPLE 5-4

Partial Amino Acid Sequence

A portion of a purified specimen of a trehalose-releasing enzyme,obtained by the method in Example 5-2, was dialyzed against distilledwater and prepared into a sample containing about 80 ng protein for theN-terminal amino acid analysis. Using “PROTEIN SEQUENCER MODEL 473A”, aprotein sequencer commercialized by Applied Biosystems, Inc., FosterCity, USA, the N-terminal amino acid sequence was analyzed up to 20amino acid residues from the N-terminus. The revealed N-terminal aminoacid sequence was the partial amino acid sequence of SEQ ID NO: 14.

A portion of a purified specimen of a trehalose-releasing enzyme,obtained by the method in Example 5-2, was dialyzed against 10 mMTris-HCl buffer (pH 9.0) and in a usual manner concentrated to give aconcentration of about one milligram per milliliter using“ULTRACENT-30”, an ultrafiltration membrane commercialized by TosohCorporation, Tokyo, Japan. To 0.2 ml of the concentrate was added 10 μgof a lysyl endopeptidase reagent commercialized by Wako Pure ChemicalIndustries, Ltd., Tokyo, Japan, and the mixture was incubated at 30° C.for 22 hours to digest the enzyme and to form peptides. The reactionmixture was subjected to reverse-phase HPLC using a column of “NOVA-PAKC18 COLUMN”, 4.5 mm in diameter and 150 mm in length, commercialized byWaters Chromatography Div., Millipore Corp., Milford, Mass., USA, toseparate the peptides under ambient temperature. The elution step wascarried out using a linear gradient of a 0.1 v/v % aqueoustrifluoroacetic acid solution containing acetonitrile increasing from 24v/v % to 48 v/v % for 60 min during the feeding at a flow rate of 0.9ml/min. Peptides eluate from the column was monitored by measuring at awavelength of 210 nm. Two peptides, named “RT18” with a retention timeof about 18 min and “RT33” with a retention time of about 33 min andwell separated from others, were collected, dried in vacuo, anddissolved respectively in a 50 v/v % aqueous acetonitrile solutioncontaining 200 μl of 0.1 v/v % trifluoroacetic acid. The peptidesolutions were subjected to a protein sequencer to analyze up to 20amino acid residues from the N-terminus of each peptide. The amino acidsequences of SEQ ID NOs: 15 and 16 from the peptides RT18 and RT33,respectively.

EXAMPLE 6

DNA Encoding Trehalose-Releasing Enzyme

EXAMPLE 6-1

Construction and Screening of Gene Library

According to Example 3-1, a gene library of Arthrobacter sp. S34, FERMBP-6450 was constructed, and then subjected to screening by applyingcolony hybridization method under the conditions as used in Example 3-1except for using as a probe an oligonucleotide, having a nucleotidesequence encoding the present trehalose-releasing enzyme, prepared bythe following procedures; The probe was in a usual manner prepared bylabelling with an isotope of [γ-³²P] ATP and T4 polynucleotide kinasethe oligonucleotide having the nucleotide sequence of SEQ ID NO: 31,which had been chemically synthesized based on an amino acid sequenceconsisting of amino acids 12-20 of SEQ ID NO: 15 revealed in Example5-4. A transformant which strongly hybridized with the prove wasselected.

According to the method in Example 3-2, a recombinant DNA was extractedfrom the transformant and analyzed on conventional Southern blottechnique using the above prove. A restriction map made based on theanalytical data was coincided with that of the recombinant DNA pGY1obtained in Examples 3-1 and 3-2. As shown in FIG. 5, it was revealedthat the present recombinant DNA in this example contained a nucleotidesequence, which encoded the present trehalose-releasing enzyme asindicated with an oblique arrow, within a region consisting of bases ofabout 2,200 bp positioned between recognition sites by restrictionenzymes, PstI and KpnI. Using the recombinant DNA pGY1, it was proceededthe decoding of the nucleotide sequence of DNA encoding the presenttrehalose-releasing enzyme.

EXAMPLE 6-2

Decoding of Nucleotide Sequence

The recombinant DNA pGY1, obtained by the method in Example 3-2, was inconventional manner completely digested with a restriction enzyme, PstI.The DNA fragment of about 3,300 bp formed in the resulting mixture wasremoved on conventional agarose electrophoresis, and the formed DNAfragment of about 5,200 bp was collected. The DNA fragment was in ausual manner subjected to ligation reaction, and the ligated product wasused to transform “XL1-BLUE”, an Escherichia coli strain commercializedby Stratagene Cloning Systems, California, USA. From the resultanttransformant, a recombinant DNA was extracted by conventional method.The recombinant DNA was confirmed to have a region consisting of basesof about 2,200 bp comprising a nucleotide sequence encoding the presenttrehalose-releasing enzyme, and named “pGZ2”. A transformantintr254oduce with pGZ2 was named a recombinant DNA pGZ2.

Analysis of Conventional dideoxy method for the nucleotide sequence ofthe recombinant DNA pGZ2 revealed that it contained a nucleotidesequence consisting of 2,218 bp bases as shown in SEQ ID NO: 32 derivedfrom Arthrobacter sp. S34, FERM BP-6450. The nucleotide sequence couldencode the amino acid sequence in SEQ ID NO: 32. The amino acid sequencewas compared with those of SEQ ID NOs: 14 to 16 as partial amino acidsequences of the present trehalose-releasing enzyme confirmed in Example5-4. As a result, the amino acid sequences of SEQ ID NOs: 14, 15 and 16were respectively coincided with amino acids 1-20, 298-317, and 31-50 ofthe amino acid sequence in SEQ ID NO: 32. The data indicates that thetrehalose-releasing enzyme in Example 5 comprises the amino acidsequence in SEQ ID NO: 32 or the one of SEQ ID NO: 9, and that theenzyme from Arthrobacter sp. S34, FERM BP-6450, is encoded by bases477-2,201 in SEQ ID NO: 32 or the nucleotide sequence of SEQ ID NO: 17.FIG. 12 shows the structure of the aforesaid recombinant DNA pGZ2.

The above amino acid sequence of the present trehalose-releasing enzyme,obtained by the method in Example 5, and other conventional ones ofenzymes having an activity of trehalose-releasing enzyme were comparedwith each other in accordance with the method in Example 3-2 todetermine their homology (%). As conventional enzymes, those derivedfrom Arthrobacter sp. Q36 disclosed in Japanese Patent Kokai No.298,880/95; Rhizobium sp. M-11, disclosed in Japanese Patent Kokai No.298,880/95; Sulfolobus acidocaldarius, ATCC 33909; and Sulfolobussolfataricus KM1 disclosed in Sai-Kohyo No. WO95/34642. All of theseenzymes have optimum temperatures out of a medium temperature range. Theamino acid sequences of these enzymes are available from the GenBank, aDNA database produced by the National Institutes of Health (NIH), USA,under the accession numbers of D63343, D64130, D78001, and D83245. Theinformation of their homology are in Table 7.

TABLE 7 Origin of enzyme for amino acid Homology on amino acidsequence(*) comparison sequence Arthrobacter sp. Q36 (D63343) 59.9%Rhizobium sp. M-11 (D78001) 59.1% Sulfolobus solfataricus KM1 37.7%(D64130) Sulfolobus acidocaldarius, 36.0% ATCC 33909 (D83245) (*):Numerals in parentheses are access numbers to the GeneBank.

As shown in Table 7, the present trehalose-releasing enzyme in Example 5showed a highest amino acid homology of 59.9% with the enzyme fromArthrobacter sp. Q36 among conventional enzymes with optimumtemperatures out of a medium temperature range. The data indicates thatthe present trehalose-releasing enzyme generally comprises an amino acidsequence with a homology of at least 60% with the amino acid sequence ofSEQ ID NO: 9. The comparison result on amino acid sequence revealed thatthe enzyme in Example 5 and the above-identified four types ofconventional enzymes have common amino acid sequences of SEQ ID NOs: 10and 13. The enzyme in Example 5 has partial amino acid sequences of SEQID NOs: 10 to 13 as found in amino acids 148-153, 185-190, 248-254 and285-291 in SEQ ID NO: 9. The four types of enzymes used as referenceshave the above partial amino acid sequences which are positioned attheir corresponding parts. Based on the fact that any of the presentenzyme in Example 5 and the enzymes as references have commonly anactivity of specifically hydrolysing a non-reducing saccharide, whichhas a trehalose structure as an end unit and a glucose polymerizationdegree of at least three, to release trehalose from the rest of rest ofthe non-reducing saccharide, it was indicated that the partial aminoacid sequences of SEQ ID NOs: 10 to 13 correlated to the expression ofsuch an enzyme activity. These results show that the presenttrehalose-releasing enzyme can be characterized in that it comprises theamino acid sequences of SEQ ID NOs: 10 to 13 and has an optimumtemperature in a medium temperature range.

EXAMPLE 6-3

Transformant Introduced with DNA

Based on the 5′- and 3′-terminal nucleotide sequences of SEQ ID NO: 17,oligonucleotides of the bases of SEQ ID NOs: 33 and 34 were chemicallysynthesized in a usual manner. As sense- and anti-sense-primers, 85 ngof each of the oligonucleotides and 100 ng of the recombinant DNA pGZ2in Example 6-2 as a template were mixed in a reaction tube while addinganother reagents in accordance with Example 3-3. The temperature for PCRwas controlled in such a manner that the mixture was treated with 25cycles of successive incubations of 95° C. for one minute, 98° C. for 20seconds, 70° C. for 30 seconds, and 72° C. for four minutes, and finallyincubated at 72° C. for 10 min. A DNA as a PCR product was collected ina usual manner to obtain an about 1,700 bp DNA. The DNA thus obtainedwas admixed with “pKK233-3”, a plasmid vector commercialized byPharmacia LKB Biotechnology AB, Uppsala, Sweden, which had beenpreviously cleaved with a restriction enzyme, EcoRI, and blunted by “DNABLUNTING KIT” commercialized by Takara Shuzo Co., Ltd., Tokyo, Japan,and ligated by conventional ligation method. Thereafter, the ligatedproduct was treated in a usual manner to obtain a recombinant DNAintroduced with the above DNA consisting of bases of about 1,700 bp.Decoding of the recombinant DNA by conventional dideoxy method showedthat it comprised a nucleotide sequence which a nucleotide sequence of5′-TGA-3′ was added to 3′-terminus of the nucleotide sequence of SEQ IDNO: 17. The DNA was named “pGZ3”. The structure of the recombinant DNApGZ3 was in FIG. 13.

The recombinant pGZ3 was in a usual manner introduced into anEscherichia coli LE 392 strain, ATCC 33572, which had been competentedin conventional manner, to obtain a transformant. Conventionalalkali-SDS method was applied for the transformant to extract a DNA andnamed “GZ3” by identifying transformant as pGZ3. Thus a transformant,introduced with the present trehalose-releasing enzyme, was obtained.

EXAMPLE 6-4

Transformant Introduced with DNA

PCR was done similarly as in Example 6-3 except for using, as sense- andanti-sense-primers, oligonucleotide having nucleotide sequences of SEQID NOs: 35 and 36, respectively, which had been chemically synthesizedbased on the 5′- and 3′-terminal nucleotide sequences of SEQ ID NO: 17.A DNA as a PCR product was collected in a usual manner to obtain anabout 1,700 bp DNA. The DNA thus obtained was cleaved with restrictionenzymes, XbaI and SpeI, and “pKK4”, a plasmid vector obtained by themethod in Example 3-4, which had been previously cleaved withrestriction enzyme, XbaI and SpeI, were ligated in a usual manner.Thereafter, the ligated product was treated in a usual manner to obtaina recombinant DNA comprising the nucleotide sequence of SEQ ID NO: 17.The recombinant DNA thus obtained was named “pKGZ1”.

A nucleotide sequence in the upper part of the 5′-terminus of SEQ ID NO:17 contained in the recombinant DNA pKGZ1 was modified similarly as inExample 3-4; PCR as a first PCR-C was carried out similarly as inExample 3-3 except for using the above recombinant DNA pKGZ1 as atemplate and oligonucleotides of SEQ ID NOs: 26 and 37, as sense- andanti-sense-primers, which had been chemically synthesized in a usualmanner based on the nucleotide sequence of the plasmid vector pKK4. Inparallel, PCR as a first PCR-D was carried out similarly as in Example3-3 except for using the above recombinant DNA pKGZ1 as a template andoligonucleotides of SEQ ID NOs: 38 and 39, as sense- andanti-sense-primers, which had been chemically synthesized in a usualmanner based on the nucleotide sequences of SEQ ID NOs: 38 and 39. A DNAas a PCR-C product was collected in a usual manner to obtain an about390 bp DNA, while another DNA as a PCR-D product was collected similarlyas above to obtain an about 590 bp DNA.

PCR as a second PCR-B was carried out similarly as in Example 3-3 exceptfor using the DNA mixture obtained as products in the first PCR-C andfirst PCR-D, an oligonucleotide of SEQ ID NO: 26 used in the first PCP-Cas a sense primer, and an oligonucleotide of SEQ ID NO: 39 used in thefirst PCR-D as an anti-sense primer. A DNA as a PCR product wascollected in a usual manner to obtain an about 950 bp DNA.

The DNA as a second PCR-B product was cleaved with a restriction enzyme,EcoRI, and the formed about 270 bp DNA was collected in conventionalmanner. The recombinant DNA pKGZ1 was cleaved with a restriction enzyme,EcoRI, and the formed about 5,100 bp DNA was collected similarly asabove. These DNAs were ligated as usual and treated in a usual manner toobtain a recombinant DNA comprising about 270 bp DNA from the secondPCR-B product. Decoding of the recombinant DNA by conventional dideoxymethod revealed that it contained the nucleotide sequence of SEQ ID NO:8, one of SEQ ID NO: 17, and one represented by 5′-TGA-3′ in the orderas indicated from the 5′- to 3′-termini. The recombinant DNA thusobtained was named “pGZ4”. The recombinant DNA pGZ4 had substantiallythe same structure as the recombinant DNA pGZ3 obtained in Example 6-3except that it had the nucleotide sequence of SEQ ID NO: 8.

The recombinant DNA pGZ4 was introduced into “BMH71-18mutS”, anEscherichia coli competent cell commercialized by Takara Shuzo Co.,Ltd., Tokyo, Japan, to obtain a transformant. Using conventionalalkali-SDS method, a DNA was extracted from the transformant andconfirmed to be pGZ4 according to conventional manner. It was named“GZ4”. Thus a transformant introduced with a DNA encoding the presenttrehalose-releasing enzyme.

EXAMPLE 7

Preparation of Trehalose-Releasing Enzyme

EXAMPLE 7-1

Preparation of Enzyme Using Microorganisms of the Genus Arthrobacter

A seed culture of Arthrobacter sp. S34, FERM BP-6450, was inoculated toa nutrient culture medium and incubated by a fermenter for about 72hours in accordance with the method in Example 2-1. After theincubation, the resultant culture was filtered and concentrated with anSF-membrane to obtain an about eight liters of cell suspension which wasthen treated with “MINI-LABO”, a super high-pressure cell disruptercommercialized by Dainippon Pharmaceutical Co., Ltd., Tokyo, Japan, todisrupt cells. The cell disruptant was centrifuged to collect and obtainan about 8.5 l supernatant as a cell extract. Determination of the cellextract for trehalose-releasing enzyme activity revealed that theculture contained about 0.3 unit/ml culture of the enzyme activity. Tothe cell extract was added ammonium sulfate to give a saturation degreeof 0.7 to effect salting out, and then centrifuged to obtain theprecipitate. The precipitate was dissolved in 10 mM phosphate buffer (pH7.0), and dialyzed against a fresh preparation of the same buffer. Thedialyzed inner solution was subjected to ion-exchange chromatographyusing “SEPABEADS FP-DA13 GEL” commercialized by Mitsubishi Chemical Co.,Ltd., Tokyo, Japan, in accordance with the method in Example 5-2 exceptthat the resin volume used of the ion exchanger was about two liters,followed by collecting fractions having an trehalose-releasing enzymeactivity. The fractions were pooled and dialyzed against a freshpreparation of the same buffer but containing 1 M ammonium sulfate, andthen the dialyzed solution was centrifuged to obtain the formedsupernatant. The supernatant was subjected to a hydrophobic columnchromatography using “BUTYL TOYOPEARL 650M GEL”, a hydrophobic gelcommercialized by Tosoh Co., Ltd., Tokyo, Japan, in accordance with themethod in Example 5-2 except that an about 350 ml of the gel was used,and then fractions with a trehalose-releasing enzyme activity werecollected. The enzyme collected was confirmed to have an optimumtemperature in a medium temperature range, i.e., temperatures over 45°C. but below 60° C. and an optimum pH in an acid pH range, i.e., a pH ofless than 7.

Thus an about 6,400 units of the present trehalose-releasing enzyme wasobtained.

EXAMPLE 7-2

Preparation of Enzyme Using Microorganism of the Genus Arthrobacter

A seed culture of Arthrobacter sp. S34, FERM BP-6450, was inoculated toa nutrient culture medium in accordance with the method in Example 7-1.To one l of the resulting culture was added 100 mg “OVALBUMIN LYSOZYME”,a lysozyme preparation, commercialized by Nagase Biochemicals, Ltd.,Kyoto, Japan. Then aeration was suspended, and cells were disrupted bykeeping the culture for 24 hours under the same temperature and stirringconditions as used in the culture. The cell disruptant was subjected toa continuous centrifuge at 10,000 rpm, followed by collecting asupernatant as a cell extract. In accordance with the method in Example7-1, the cell extract was treated with salting out, and the sediment wasdialyzed. The resulting dialyzed inner solution was subjected toion-exchange chromatography using “SEPABEADS FP-DA13 GEL”, a product ofMitsubishi Chemical Co., Ltd., Tokyo, Japan, in accordance with themethod in Example 7-1 to collect fractions with a trehalose-releasingenzyme activity. The pooled fractions contained about 16,500 units ofthe present trehalose-releasing enzyme and about 5,500 units the presentnon-reducing saccharide-forming enzyme. Thus an enzyme preparationcontaining the present two types of enzymes was obtained.

EXAMPLE 7-3

Production of Enzyme Using Transformant

In a 500-ml Erlenmeyer flask were placed a 100 ml aqueous solutioncontaining 16 g/l polypeptone, 10 g/l yeast extract, and 5 g/l sodiumchloride, and the flask was autoclaved at 121° C. for 15 min, cooled,aseptically adjusted to pH 7.0, and aseptically admixed with 10 mgampicillin in a sodium salt to obtained a nutrient culture medium. Thetransformant “GZ2” obtained in Example 6-2 was inoculated into theliquid medium, followed by the incubation at 37° C. for about 20 hoursunder aeration-agitation conditions to obtain a seed culture. Sevenliters of a fresh preparation of the same medium as used in the seedculture were similarly prepared and placed in a 10-l fermenter,inoculated with 70 ml of the seed culture, and cultures for about 20hours under aeration-agitation conditions. Cells were collected bycentrifuging the resulting culture in usual mariner. The collected cellswere suspended in 10 mM phosphate buffer (pH 7.0) and ultrasonicated todisrupt the cells. The resulting mixture was centrifuged to removeinsoluble substances, followed by collecting a supernatant as a cellextract. The cell extract was dialyzed against 10 mM phosphate buffer(pH 7.0). The dialyzed inner solution was collected and confirmed tohave an optimum temperature in a medium temperature range, i.e.,temperatures over 45° C. but below 60° C. and an optimum pH in an acidpH range, i.e., a pH of less than 7.

Thus the present trehalose-releasing enzyme was obtained. In thisExample, an about 0.5 unit/ml culture of the trehalose-releasing enzymewas obtained.

As a control, “XL1-Blue”, an Escherichia Coli strain commercialized byStratagene Cloning Systems, California, USA, was cultured under the sameculture conditions as used in the above in a fresh preparation of thesame culture medium as above but free of ampicillin, followed bycollecting and dialyzing a cell extract similarly as above. Notrehalose-releasing enzyme activity was observed, meaning that thetransformant GZ2 can be advantageously used in producing the presenttrehalose-releasing enzyme.

EXAMPLE 7-4

Production of Enzyme Using Transformant

The transformant GZ3 in Example 6-3 was cultured similarly as in Example7-3 except for using a liquid nutrient culture medium (pH 7.0)consisting of one w/v % maltose, three w/v % polypeptone, one w/v %“MEAST PIG” commercialized by Asahi Breweries, Ltd., Tokyo, Japan, 0.1w/v % dipotassium hydrogen phosphate, 100 μg/ml ampicillin, and water.The resulting culture was treated with ultrasonication to disrupt cells,and the mixture was centrifuged to remove insoluble substances.Measurement of the trehalose-releasing enzyme activity in the resultingsupernatant revealed that it contained about 70 units/ml culture of theenzyme. In accordance with the method in Example 5-2, the supernatantwas purified and confirmed that the purified specimen had an optimumtemperature in a medium temperature range, i.e., temperatures over 45°C. but below 60° C. and an optimum pH in an acid pH range, i.e., a pH ofless than 7. Thus the present trehalose-releasing enzyme was obtained.

EXAMPLE 7-5

Production of Enzyme Using Transformant

The transformant GZ4 in Example 6-4 was cultured similarly as in Example4-4. The resulting culture was treated with ultrasonication to disruptcells, and the mixture was centrifuged to remove insoluble substances.Measurement of the trehalose-releasing enzyme activity in the resultingsupernatant revealed that it contained about 250 units/ml culture of theenzyme. In accordance with the method in Example 5-2, the supernatantwas purified and confirmed that the purified specimen had an optimumtemperature in a medium temperature range, i.e., temperatures over 45°C. but below 60° C. and an optimum pH in an acid pH range, i.e., a pH ofless than 7. Thus the present trehalose-releasing enzyme was obtained.

EXAMPLE 8

Saccharide Production

EXAMPLE 8-1

Production of Non-Reducing Saccharide Syrup

A 6 w/w % potato starch suspension was gelatinized by heating, adjustedto pH 4.5 and 50° C., admixed with 2,500 units/g starch, d.s.b., andenzymatically reacted for 20 hours. The reaction mixture was adjusted topH 6.5, autoclaved at 120° C. for 10 min, cooled to 40° C., admixed with150 units/g starch, d.s.b., of “TERMAMYL 60L”, an α-amylase specimencommercialized by Novo Nordisk Industri A/S, Copenhagen, Denmark, andsubjected to an enzymatic reaction for 20 hours while keeping at thetemperature. The reaction mixture was autoclaved at 120° C. for 20 min,cooled to 53° C., adjusted to pH 5.7, admixed with one unit per gramstarch, d.s.b., of a non-reducing saccharide-forming enzyme obtained bythe method in Example 4-1, and subjected to an enzymatic reaction for 96hours. The reaction mixture thus obtained was heated at 97° C. for 30min to inactivate the remaining enzyme, cooled, filtered, purified in ausual manner by decoloration with an activated charcoal and desaltingwith ion exchangers, and concentrated to obtain an about 70 w/w % syrupin a yield of about 90% to the material starch, d.s.b.

The product, which has a low DE of 24 and contains α-glucosyltrehalose,α-maltosyltrehalose, α-maltotriosyltrehalose, α-maltotetraosyltrehalose,and α-maltopentaosyltrehalose in respective amount of 11.5, 5.7, 29.5,3.5, and 2.8%, d.s.b., has a mild and high-quality sweetness, and asatisfactory viscosity and moisture-retaining ability. It can bearbitrarily used as a sweetener, taste-improving agent,quality-improving agent, stabilizer, filler, adjuvant or excipient incompositions in general such as foods, cosmetics, and pharmaceuticals.

EXAMPLE 8-2

Production of Syrup Containing Non-Reducing Saccharide

To a 33 w/w % corn starch suspension was added calcium carbonate to givea final concentration of 0.1 w/w %, and then the mixture was adjusted topH 6.5, admixed with 0.2 w/w % per starch, d.s.b., of “TERMAMYL 60L”, aliquefying α-amylase specimen commercialized by Novo Nordisk IndustriA/S, Copenhagen, Denmark, and enzymatically reacted at 95° C. for 15 minto liquefy the starch. The liquefied starch was autoclaved at 120° C.for 10 min, cooled to 53° C., admixed with one unit/g starch, d.s.b., ofa maltotetraose-forming enzyme from a Pseudomonas stutzeri straincommercialized by Hayashibara Biochemical Laboratories Inc., Okayama,Japan, and two units/g starch, d.s.b., of a non-reducingsaccharide-forming enzyme obtained by the method in Example 4-2, andenzymatically reacted for 48 hours. The reaction mixture was admixedwith 15 units of “α-AMYLASE 2A”, an α-amylase specimen commercialized byUeda Chemical Co., Ltd., Osaka, Japan, and then incubated at 65° C. fortwo hours, autoclaved at 120° C. for 10 min, and cooled. The resultingmixture was filtered, and in a usual manner purified by treatments ofcoloration using an activated charcoal and of desalting using ionexchangers, and concentrated into an about 70 w/w % syrup, d.s.b., in ayield of about 90% with respect to the material starch, d.s.b.

The product, which has a low DE of 18.5 and containsα-glucosyltrehalose, α-maltosyltrehalose, α-maltotriosyltrehalose,α-maltotetraosyltrehalose, and α-maltopentaosyltrehalose in respectiveamount of 9.3, 30.1, 0.9, 0.8, and 0.5%, d.s.b., has a mild andhigh-quality sweetness, and a satisfactory viscosity andmoisture-retaining ability. It can be arbitrarily used as a sweetener,taste-improving agent, quality-improving agent, stabilizer, filler,adjuvant or excipient in compositions in general such as foods,cosmetics, and pharmaceuticals.

EXAMPLE 8-3

Production of Non-Reducing Saccharide

A 20 w/w % aqueous solution of any of reducing partial starchhydrolyzates of maltotriose, maltotetraose, maltopentaose, maltohexaose,and maltoheptaose, which are all produced by Hayashibara BiochemicalLaboratories Inc., Okayama, Japan, admixed with two units/g reducingpartial starch hydrolyzate of a purified specimen of non-reducingsaccharide-forming enzyme obtained by the method in Example 2-2, andsubjected to an enzymatic reaction at 50° C. and pH 6.0 for 48 hours.From each of the above-identified reducing partial starch hydrolyzateswere respectively formed α-glucosyltrehalose, α-maltosyltrehalose,α-maltotriosyltrehalose, α-maltotetraosyltrehalose, andα-maltopentaosyltrehalose as reducing saccharides. Saccharides in eachreaction mixture were in conventional manner fractionated by thefollowing successive treatments: Inactivation of the remaining enzyme byheating, filtration, decoloration, desalting, concentration, and columnchromatography using “XT-1016 (Na⁺-form)”, an alkali-metal strong-acidcation exchange resin with a polymerization degree of 4%, commercializedby Tokyo Organic Chemical Industries, Ltd., Tokyo, Japan. The conditionsused in the column chromatography were as follows: The inner columntemperature was set to 55° C., the load volume of a saccharide solutionto the resin was about 5 v/v %, and the flow rate of water heated to 55°C. as a moving bed was set to SV (space velocity) 0.13. An eluate fromeach column, which contained at least 95 w/w % of any of theabove-identified non-reducing saccharides, d.s.b., with respect tosaccharide composition, was collected. To each collected eluate wasadded sodium hydroxide to give a concentration of 0.1 N, and the mixturewas heated at 100° C. for two hours to decompose the remaining reducingsaccharides. The reaction mixtures thus obtained were respectivelydecolored with an activated charcoal, desalted with ion exchangers in H-and OH-form, concentrated, dried in vacuo, and pulverized into powderyα-glucosyltrehalose, α-maltosyltrehalose, α-maltotriosyltrehalose,α-maltotetraosyltrehalose, and α-maltopentaosyltrehalose with a purityof at least 99.0 w/w %, d.s.b.

The products, containing highly-purified non-reducing saccharides andhaving a more lower DE, can be arbitrarily used as a taste-improvingagent, quality-improving agent, stabilizer, filler, adjuvant orexcipient in compositions in general such as foods, cosmetics, andpharmaceuticals.

EXAMPLE 8-4

Production of Crystalline Powder Containing Non-Reducing Saccharide

An aqueous 20 w/w % solution of maltopentaose commercialized byHayashibara Biochemical Laboratories Inc., Okayama, Japan, was prepared,admixed with two units/g maltopentaose, d.s.b., of a non-reducingsaccharide-forming enzyme obtained by the method in Example 4-3, andenzymatically reacted at 50° C. for 48 hours, resulting in a conversionof about 75% maltopentaose into α-maltotriosyltrehalose. The reactionmixture was heated at 97° C. for 30 min to inactivate the remainingenzyme, and then cooled, filtered, and purified by decoloration using anactivated charcoal and desalting using ion exchangers.

Thereafter, the resulting solution was concentrated into an about 75 w/w% solution with respect to solid contents, admixed with an about 0.01w/v α-maltotriosyltrehalose crystal as a seed crystal, and allowed tostand for 24 hours. Then the crystallized α-maltotriosyltrehalosecrystal was collected by a centrifuge, washed with a small amount ofcold water, and dried in a usual manner to obtain a crystalline powderwith a relatively-high content of the non-reducing saccharide in a yieldof about 50% to the material solids, d.s.b.

The product, having a relatively-low sweetness and an extremely-low DEof less than 0.2 and containing at least 99.0 w/w % ofα-maltotriosyltrehalose as a non-reducing saccharide, can be arbitrarilyused as a taste-improving agent, quality-improving agent, stabilizer,filler, adjuvant or excipient in compositions in general such as foods,cosmetics, and pharmaceuticals.

EXAMPLE 8-5

Process for Producing Hydrous Crystalline Trehalose

Corn starch was suspended in water into a 30 w/w % starch suspensionwhich was then admixed with calcium carbonate in an amount of 0.1 w/w %.The mixture was adjusted to pH 6.0, and then admixed with 0.2 w/w % perstarch, d.s.b., of “TERMAMYL 60L”, a liquefying α-amylase specimencommercialized by Novo Nordisk Industri A/S, Copenhagen, Denmark, andenzymatically reacted at 95° C. for 15 min to gelatinize and liquefy thestarch. The resulting mixture was autoclaved at 120° C. for 30 min,cooled to 51° C., adjusted to pH 5.7, and enzymatically reacted at thesame temperature for 64 hours after admixed with 300 units/g starch,d.s.b., of an isoamylase specimen commercialized by HayashibaraBiochemical Laboratories Inc., Okayama, Japan; two units/g starch,d.s.b., of a cyclomaltodextrin glucanotransferase specimencommercialized by Hayashibara Biochemical Laboratories Inc., Okayama,Japan; two units of a non-reducing saccharide-forming enzyme obtained bythe method in Example 4-1; and 10 unit/g starch, d.s.b., of atrehalose-releasing enzyme obtained by the method in Example 7-1. Thereaction mixture was heated at 97° C. for 30 min to inactivate theremaining enzyme, and then cooled 50° C., admixed with 10 unit/g starch,d.s.b., of “GLUCOZYME”, a glucoamylase specimen commercialized by NagaseBiochemicals, Ltd., Kyoto, Japan, and subjected to an enzymatic reactionfor 24 hours. The reaction mixture thus obtained was heated at 95° C.for 10 min to inactivate the remaining enzymes, cooled, filtered,purified by decoloration using an activated charcoal and desalting usingion exchangers, and concentrated to an about 60 w/w % solution withrespect to solid contents or a syrup containing 84.1 w/w % trehalose,d.s.b. The syrup was concentrated up to give a concentration of about 83w/w %, d.s.b., and the concentrate was placed in a crystallizer, admixedwith an about 0.1 w/v % hydrous crystalline trehalose to the syrup, andstirred for about two hours to crystallize the saccharide. The resultingcrystals were collected by a centrifuge, washed with a small amount ofwater to remove molasses, dried by air heated to 45° C. to obtainhydrous crystalline trehalose with a purity of at least 99% in a yieldof about 50% to the material starch, d.s.b.

Since the product is substantially free from hygroscopicity and easilyhandleable, it can be arbitrarily used as a sweetener, taste-improvingagent, quality-improving agent, stabilizer, filler, adjuvant orexcipient in compositions in general such as foods, cosmetics, andpharmaceuticals.

EXAMPLE 8-6

Process for Producing Crystalline Powder Containing AnhydrousCrystalline Trehalose

Using the method in Example 8-5 hydrous crystalline trehalose wasprepared, and the saccharide was dried in vacuo using a jacketedrotary-vacuum-dryer. The drying was conducted at 90° C. and 300-350 mmHgfor about seven hours. After the drying, the above temperature andpressure were returned to ambient temperature and normal pressure beforecollecting the product or a crystalline powder containing at least 90w/w % anhydrous crystalline trehalose, d.s.b.

Since anhydrous crystalline trehalose absorbs moisture in hydrousmatters and changes in itself into hydrous crystalline trehalose, theproduct rich in the saccharide can be arbitrarily used as a non-harmfulsafe desiccant to dehydrate or dry compositions including food products,cosmetics and pharmaceuticals, as well as materials and intermediatesthereof. The product with a mild and high-quality sweetness can bearbitrarily used as a sweetener, taste-improving agent,quality-improving agent, stabilizer, filler, adjuvant or excipient incompositions in general such as foods, cosmetics, and pharmaceuticals.

EXAMPLE 8-7

Process for Producing Trehalose Syrup

A 27 w/w % suspension of tapioca starch was admixed with calciumcarbonate to give a final concentration of 0.1 w/w %, adjusted to pH6.0, admixed with 0.2 w/w % per starch, d.s.b., of “TERMAMYL 60L”, aliquefying α-amylase specimen commercialized by Novo Nordisk IndustriA/S, Copenhagen, Denmark, and enzymatically reacted at 95° C. for 15 minto gelatinize and liquefy the starch. The resulting mixture wasautoclaved at a pressure of 2 kg/cm² for 30 min, cooled to 53° C.,adjusted to pH 5.7, and enzymatically reacted at the same temperaturefor 72 hours after admixed with 500 units/g starch, d.s.b., of“PROMOZYME 200L”, a pullulanase specimen commercialized by Novo NordiskIndustri A/S, Copenhagen, Denmark; one unit/g starch, d.s.b., ofPseudomonas stutzeri strain commercialized by Hayashibara BiochemicalLaboratories Inc., Okayama, Japan; about two units/g starch, d.s.b., ofa non-reducing saccharide-forming enzyme and about six units/g starch,d.s.b., of a trehalose-releasing enzyme, obtained by the method inExample 7-2. The reaction mixture thus obtained was heated at 97° C. for15 min, cooled and filtered to obtain a filtrate. The filtrate was in ausual manner purified by decoloration using an activated charcoal anddesalting using ion exchangers, and concentrated to an about 70 w/w %syrup with respect to solid contents in a yield of about 92% to thematerial, d.s.b.

The product, comprising 35.2% trehalose, 3.4% α-glucosyltrehalose, 1.8%glucose, 37.2% maltose, 9.1% maltotriose, and 13.3% oligosaccharideshigher than maltotetraose, has a mild and high-quality sweetness,relatively-lower reducibility and viscosity, and adequatemoisture-retaining ability; it can be arbitrarily used as a sweetener,taste-improving agent, quality-improving agent, stabilizer, filler,adjuvant or excipient in compositions in general such as foods,cosmetics, and pharmaceuticals.

EXAMPLE 8-8

Process for Producing Crystalline Powder Containing AnhydrousCrystalline Trehalose

One part by weight of “EX-I”, an amylose commercialized by HayashibaraBiochemical Laboratories Inc., Okayama, Japan, was dissolved in 15 partsby weight of water by heating, and the solution was heated to 53° C. andadjusted to pH 5.7. To the resulting solution was added two units/gamylose, d.s.b., of a non-reducing saccharide-forming enzyme, obtainedin Example 4-3, and six units/g amylose, d.s.b., of atrehalose-releasing enzyme, obtained by the method in Example 7-4,followed by an incubation for 48 hours. The reaction mixture was heatedat 97° C. for 30 min to inactivate the remaining enzyme, and thenadjusted to pH 5.0, admixed with 10 units/g amylose, d.s.b., of“GLUCOZYME”, a glucoamylase specimen commercialized by NagaseBiochemicals, Ltd., Kyoto, and enzymatically reacted for 40 hours. Thereaction mixture thus obtained was heated at 95° C. for 10 min toinactivate the remaining enzymes, cooled, filtered, purified bydecoloration using an activated charcoal and desalting using ionexchangers, and concentrated to an about 60 w/w % solution with respectto solid contents or a syrup containing 82.1 w/w % trehalose, d.s.b.

Similarly as in Example 8-3, the syrup was subjected to columnchromatography, followed by collecting a fraction containing about 98w/w % trehalose, d.s.b. The fraction was concentrated in vacuo underheating conditions into an about 85 w/w % syrup with respect to solidcontents. The syrup was admixed with hydrous crystalline trehalose as aseed crystal in an about 2 w/v % of to the syrup, stirred at 120° C. forfive minutes, distributed to plastic vats, and dried at 100° C. in vacuoto crystallize the saccharide. Thereafter, the contents in a block formwere detached from the vats and cut with a cutter to obtain a solidproduct, containing anhydrous crystalline trehalose with a crystallinityof about 70% and having a moisture content of about 0.3 w/w % in a yieldof about 70% to the material amylose, d.s.b. The solid product waspulverized in a usual manner into a crystalline powdery containinganhydrous crystalline trehalose.

Since anhydrous crystalline trehalose absorb moisture from hydrousmatters and changes into hydrous crystalline trehalose, the product richin anhydrous crystalline trehalose can be arbitrarily used as anon-harmful safe desiccant to dehydrate or dry compositions includingfood products, cosmetics and pharmaceuticals, as well as materials andintermediates thereof. The product with a mild and high-qualitysweetness can be arbitrarily used as a sweetener, taste-improving agent,quality-improving agent, stabilizer, filler, adjuvant or excipient incompositions in general such as foods, cosmetics, and pharmaceuticals.

As described above, the present invention was made based on the findingof a novel non-reducing saccharide-forming enzyme and a noveltrehalose-releasing enzyme, which have an optimum temperature in amedium temperature range and preferably have an optimum pH in an acid pHrange. These enzymes according to the present invention can be obtainedin a desired amount, for example, by culturing microorganisms capable ofproducing the enzymes. The present DNAs which encode either of theenzymes are quite useful in producing such enzymes as recombinantproteins. In cases of using transformant introduced with the DNAs, theenzymes according to the present invention can be yielded in a desiredamount. The present enzymes can be used in producing non-reducingsaccharides having a trehalose structure, which include trehalose, in amedium temperature rang and/or an acid pH range. Particularly, when usedthe present enzymes in combination with other saccharide-related enzymeshaving an optimum temperature in a medium temperature rang and/or anoptimum pH in an acid pH range, desired saccharides can be producedquite efficiently. The enzymes according to the present invention areones with revealed amino acid sequences; they can be safely used toproduce the non-reducing saccharides to be used in food products andpharmaceuticals. The non-reducing saccharides and reducing saccharides,which contain the same and have a lesser reducibility, produced by thepresent invention have a mild and high-quality sweetness, and mostpreferably have an insubstantial reducibility or a reduced reducibilityby a large margin. Therefore, the saccharides can be arbitrarily used asin compositions in general such as foods, cosmetics, and pharmaceuticalswith lesser fear of coloration and deterioration.

The present invention with these unfathomable advantageous propertiesand features is a useful invention that would greatly contribute to thisart.

While there has been described what is at present considered to be thepreferred embodiments of the invention, it will be understood thatvarious modifications may be made therein, and it is intended to coverin the appended claims all such modifications as fall within the truespirit and scope of the invention.

1. A process for producing a non-reducing saccharide having a trehalosestructure as an end unit, trehalose, or a saccharide compositioncomprising the same, comprising: subjecting a reducing partial starchhydrolysate to the action of (1) a non-reducing saccharide-formingenzyme which forms said non-reducing saccharide from a reducing partialstarch hydrolysate, and has an optimum temperature of over 40° C. butbelow 60° C. to produce a non-reducing saccharide, wherein saidnon-reducing saccharide-forming enzyme comprises the amino acid sequenceof SEQ ID NO: 1, consist of the amino acid sequence of SEQ ID NO: 1, andoptionally (2) a trehalose-releasing enzyme which hydrolyzes thenon-reducing saccharide having a trehalose structure as an end unit torelease said trehalose from the rest of said non-reducing saccharide,and has an optimum temperature of over 45° C. but below 60° C. toproduce trehalose, wherein said trehalose-releasing enzyme comprises theamino acid sequence of SEQ ID NO: 9 or consists of the amino acidsequence of SEQ ID NO: 9; and collecting the produced non-reducingsaccharide, trehalose, or a saccharide composition comprising the samefrom the resulting reaction mixture.
 2. The process of claim 1, whereinsaid non-reducing saccharide-forming enzyme has the followingphysicochemical properties: (1) Action Forming a non-reducing saccharidehaving a trehalose structure as an end unit from a reducing partialstarch hydrolysate having a degree of glucose polymerization of 3 orhigher; (2) Molecular weight About 75,000±10,000 daltons on sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); (3)Isoelectric point (pI) About 4.5±0.5 on isoelectrophoresis usingampholyte; (4) Optimum temperature About 50° C. when incubated at pH 6.0for 60 min; (5) Optimum pH About 6.0 when incubated at 50° C. for 60min; (6) Thermal stability Stable up to a temperature of about 55° C.when incubated at pH 7.0 for 60 min; and (7) pH stability Stable at pHsof about 5.0 to about 10.0 when incubated at 4° C. for 24 hours.
 3. Theprocess of claim 1, wherein said trehalose-releasing enzyme has thefollowing physicochemical properties: (1) Action Hydrolyzing anon-reducing saccharide having a trehalose structure as an end unit torelease said trehalose from the rest of said non-reducing saccharide;(2) Molecular weight About 62,000±5,000 daltons on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE); (3) Optimumtemperature About 50° C. to about 55° C. when incubated at pH 6.0 for 30min; (4) Optimum pH About 6.0 when incubated at 50° C. for 30 min; (5)Thermal stability Stable up to a temperature of about 50° C. whenincubated at pH 7.0 for 60 min; and (6) pH stability Stable at pHs ofabout 4.5 to about 10.0 when incubated at 4° C. for 24 hours.
 4. Theprocess of claim 1, wherein said reducing starch hydrolysate is onehaving a glucose polymerization degree of 3 or higher and beingobtainable by subjecting starch or amylaceous substance to the action ofan acid and/or starch hydrolase.
 5. The process of claim 1, wherein, inthe subjecting step, one or more enzymes selected from the groupconsisting of α-amylase, β-amylase, glucoamylase, starch-debranchingenzyme, cyclomaltodextrin glucanotransferase, and α-glucosidase arefurther allowed to act on the reducing partial starch hydrolysate. 6.The process of claim 1, wherein said trehalose is further crystallizedto produce hydrous- or anhydrous-crystalline trehalose.
 7. The processof claim 1, wherein said non-reducing saccharide having a trehalosestructure as an end unit is one or more members selected from the groupconsisting of α-glucosyltrehalose, α-maltosyltrehalose,α-maltotriosyltrehalose, α-maltotetraosyltrehalose, andα-maltopentaosyltrehalose.